Polyhydroxyalkanoate production methods and materials and microorganisms used in same

ABSTRACT

Embodiments of the invention relate generally to methods to generate microorganisms and/or microorganism cultures that exhibit the ability to produce polyhydroxyalkanoates (PHA) from carbon sources at high efficiencies. In several embodiments, preferential expression of, or preferential growth of microorganisms utilizing certain metabolic pathways, enables the high efficiency PHA production from carbon-containing gases or materials. Several embodiments relate to the microorganism cultures, and/or microorganisms isolated therefrom.

RELATED CASES

This application is a continuation of co-pending U.S. application Ser.No. 15/643,905, filed Jul. 7, 2017, which is a continuation of U.S.application Ser. No. 14/740,056, filed Jun. 15, 2015 (now issued as U.S.Pat. No. 9,725,744), which is a continuation of U.S. application Ser.No. 13/802,622, filed Mar. 13, 2013 (now issued as U.S. Pat. No.9,085,784), which claims the benefit of U.S. Provisional Application No.61/617,534, filed on Mar. 29, 2012 the entire disclosure of each ofwhich is incorporated in its entirety by reference herein.

BACKGROUND Field of the Invention

Embodiments of the invention relate to an improved process for theproduction, processing, and functional modification ofpolyhydroxyalkanoates (PHAs), and specifically to processes for theproduction, processing, and functional modification of PHAs, whereinthose PHAs may be made from carbon-containing gases and materials.

Description of the Related Art

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters that serve ascarbon and energy storage vehicles in microorganisms. PHAs are naturallybiodegradable in both aerobic and anaerobic conditions, arebiocompatible with mammalian tissues, and, as thermoplastics, can beused as alternatives to fossil fuel-based synthetic plastics such aspolypropylene, polyethylene, and polystyrene. In comparison totraditional petrochemical-based plastics, which are neitherbiodegradable nor made from sustainable sources of carbon, PHA plasticsafford significant environmental benefits.

The utilization of food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems is often regarded asthe most efficient and economical platform for PHA production.Specifically, sugar-based PHA production processes are capable ofgenerating high density fermentation cultures and high PHA inclusionconcentrations, and, by maximizing the cell culture density and PHAinclusion concentration therein, it is believed that carbon, chemical,and energy efficiencies are also maximized. For example, comparing a lowcell and PHA concentration process to a high cell and PHA concentrationprocess, a low concentration process requires significantly more, pergiven unit of PHA-containing biomass, i) energy for dewatering cellsprior to PHA extraction treatment, ii) liquid culture volume, andassociated chemicals, mixing energy, and heat removal energy, and iii)both energy and chemicals for separating PHA from biomass. Accordingly,whereas the sugar-based genetically-engineered microorganism PHA processyields maximized cell densities and PHA concentrations relative to lowconcentration processes, it is also regarded as the most carbon,chemical, energy, and, thus, cost efficient PHA production method.

Unfortunately, despite these maximized efficiency advantages,sugar-based PHA production remains more expensive than fossil fuel-basedplastics production. Thus, given the apparent efficiency maximization ofthe high density sugar-derived PHA production process, PHAs aregenerally considered to be unable to compete with fossil fuel-basedplastics on energy, chemical, and cost efficiency.

SUMMARY

Despite the environmental advantages of PHAs, the high cost of PHAproduction relative to the low cost of fossil fuel-based plasticsproduction has significantly limited the industrial production andcommercial adoption of PHAs.

To reduce the carbon input cost of the PHA production process,carbon-containing industrial off-gases, such as carbon dioxide, methane,and volatile organic compounds, have been proposed as an alternative tofood crop-based and plant-based sources of carbon. In addition to thewide availability and low cost of carbon-containing gases,carbon-containing gases also do not present the environmental challengesassociated with food crop and plant-derived sources of carbon.Specifically, whereas food crop- and plant-based carbon substratesrequire land, fertilizers, pesticides, and fossil fuels to produce, andalso generate greenhouse gas emissions during the course of production,carbon-containing off-gases do not require new inputs of land,fertilizers, or pesticides to generate. Thus, on both an economic andenvironmental basis, the utilization of carbon emissions for theproduction of PHA would appear to offer significant advantages oversugar-based PHA production processes.

Unfortunately, the fermentation or biotechnological conversion ofcarbon-containing gases into PHAs presents technical challenges andstoichiometric limitations that have, in the past, rendered thegas-to-PHA production process significantly more energy and chemicalintensive, and thus more costly, than the food crop-based PHA productionprocess. These technical challenges and stoichiometric limitationsinclude, but are not limited to low mass transfer rates, lowmicroorganism growth rates, extended polymerization times, low celldensities, high oxygen demand, low PHA cellular inclusionconcentrations, low polymer production per unit of biocatalystproduction, low biocatalyst yield per unit gas input, poor polymerfunctionality, and/or high downstream functionalization costs. Whereassugar-based fermentation or bioconversion systems have the ability togenerate high cellular densities and PHA inclusion concentrations, basedon cell morphology and mass transfer constraints, carbon-containinggas-based fermentation or bioconversion processes typically generate10-70% of the biomass and intracellular PHA inclusion concentrationsachieved in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon mass transfer,oxygen mass transfer, and culture mixing, as well as downstream PHAprocessing and/or purification, significantly exceeds the energy-to-PHAratio required for sugar-based PHA production methods, thereby renderingthe emissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based PHAs.

In light of the potential environmental advantages and carbon costefficiencies of utilizing carbon-containing gases as a source of carbonfor PHA production, there exists a significant need to reduce theenergy, chemical, and carbon input-to-PHA output ratio in a carbonemissions-based PHA production system, and thereby render carbongas-derived PHA economically competitive with petrochemical-basedplastics.

Thus, in several embodiments, there is provided a methanotrophicmicroorganism (or a culture of such microorganisms) that are capable ofgenerating PHA, particularly at high efficiency levels, therebyresulting in increased PHA production yield (e.g., high PHAconcentrations per unit mass of microorganism or unit weight ofculture).

In several embodiments, there is provided a methanotrophic microorganismcharacterized by a lack of the genetic material encoding soluble methanemonooxygenase (sMMO), presence of genetic material encoding theethylmalonyl-CoA pathway, transcription of DNA encoding theethylmalonyl-CoA pathway, and translation of mRNA encoding theethylmalonyl-CoA pathway and, the capability of producingpolyhydroxyalkanoate (PHA).

In several embodiments, the methanotrophic microorganism possessesgenetic material encoding the sMMO enzyme, but fails to eithertranscribe DNA encoding sMMO or translate mRNA encoding sMMO, and/orfails to produce a functional sMMO enzyme.

In several embodiments, the methanotrophic microorganism is from a genusselected from a group consisting of: Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas. In severalembodiments, the methanotrophic microorganism is derived from amethanotrophic microorganism of one of such genera.

In several embodiments, the methanotrophic microorganism exhibitsparticulate methane monooxygenase activity in the presence of copper ionconcentrations between 0.001 micromolar and 1000 micromolar, while insome embodiments the methanotrophic microorganism exhibitsethylmalonyl-CoA pathway activity in the presence of copperconcentrations between 0.001 micromolar and 1000 micromolar. In severalembodiments, both particulate methane monooxygenase activity andethylmalonyl-CoA pathway activity are exhibited in the same or similarconditions. In several embodiments, the methanotrophic microorganismexhibits little or no soluble methane monooxygenase activity under thesame or similar conditions.

In several embodiments, the methanotrophic microorganism obtains therecited characteristics due to one or more of mutation,genetically-engineered mutation, and/or selection-pressure-inducedmutation. Selection-pressure-induced mutation, as used herein, shall begiven its ordinary meaning and shall also refer to conditions in whichthe methanotrophic microorganism is cultured that favor one or moremicroorganisms having certain characteristics. Depending on theembodiments, this may be, for example, ability to upregulate or utilizea particular biochemical or metabolic pathway in response to one or morevariables in the culture environment (e.g., nutrients, populationdensity, pH, temperature, salinity, etc.). In several embodiments, theselection-pressure-induced mutation shall also refer to conditions thatexploit certain competitive advantages possessed by certainmethanotrophic microorganisms and not by others (or possessed orexploited to a lesser extent).

In several embodiments, the methanotrophic microorganism is capable ofproducing PHA at intracellular PHA concentrations with a ratio of PHA tonon-PHA biomass exceeding 3:1 on a dry weight basis. In severalembodiments, the methanotrophic microorganism are capable of producingPHA at a concentration (based on dry weight of a microbial culture ofthe methanotrophic microorganisms of at least about 51%. In severalembodiments, the PHA is produced at least about 71%, or at least about81% of the total dry cell weight of the methanotrophic microorganisms.

In several embodiments, there is provided an isolated or purifiedculture of microorganisms having the characteristics described herein.

In several embodiments, there is also provided a method for enhanced PHAproduction by a culture of methanotrophic microorganisms, comprising,(a) providing a culture medium comprising methane, copper, and at leastone additional nutrient, (b) providing a culture comprisingmethanotrophic microorganisms, (c) culturing the methanotrophicmicroorganisms in the culture medium, (d) controlling the concentrationof copper in the medium to result in a copper concentration suitable formethanotrophic microorganisms to produce soluble methane monooxygenase(sMMO), (e) reducing, for a first period of time, the concentration ofthe at least one additional nutrient in the medium to cause themethanotrophic microorganisms to produce PHA, (f) increasing, for asecond period of time, the concentration of the at least one additionalnutrient of step (e) to cause the methanotrophic microorganisms toreproduce using essentially only particulate methane monooxygenase(pMMO), and (g) subjecting the culture of methanotrophic microorganismsto at least two repetitions of steps (d), (e), and (f). In severalembodiments, the at least two repetitions result in the culture ofmethanotrophic microorganisms producing PHA at concentrations exceedingthose produced absent the at least two repetitions.

In several embodiments, the culturing is performed under non-sterileconditions, though PHA production can also be performed, optionally, insterile or semi-sterile conditions.

In several embodiments, PHA is produced at concentrations are at leastabout 51% of total dry cell weight of the methanotrophic microorganisms.In several embodiments the PHA produced is at least about 71% of thetotal dry cell weight of the methanotrophic microorganisms, while instill additional embodiments, the PHA produced is at least about 81% oftotal dry cell weight of the methanotrophic microorganisms

In several embodiments, the copper concentration is controlled (e.g.,increased, decreased, held etc.) to be between about 0.001 micromolarand about 1000 micromolar.

In several embodiments, the first period of time ranges from about 2 to24 hours, including about 2 to about 6, about 6 to about 10, about 10 toabout 14, about 14 to about 16, about 16 to about 20, about 20 to about24 hours, and overlapping ranges thereof. In several embodiments, thesecond period of time ranges from about 12 to 24 hours, including about12 to about 16, about 16 to about 20, about 20 to about 24, andoverlapping ranges thereof. Longer or shorter time periods may be usedfor the first period of time, the second period of time, and/or for boththe first and the second period of time, depending on the embodiment.

In several embodiments, the additional nutrient comprises at least oneof the nutrients selected from the group consisting of aluminum, boron,calcium, carbon, carbon dioxide, cobalt, iron, magnesium, molybdenum,nitrogen, oxygen, phosphorus, potassium, sodium, and zinc. In oneembodiment, the at least one additional nutrient comprises dissolvedoxygen and the method further comprises increasing the concentration ofdissolved oxygen in the culture media to preferentially select formethanotrophic microorganisms exhibiting reduced pigmentation.

In several embodiments, the at least two repetitions induce most orsubstantially all of the methanotrophic microorganisms to express pMMOand fail to express sMMO, contain the genetic material for sMMO, and/orproduce a functional sMMO.

Additionally, in several embodiments, the present invention relates tonovel processes for the conversion of carbon-containing gases into PHAsat previously unattainable energy and carbon PHA conversion ratios.Moreover, there are provided herein, systems, methods and materials forthe production and processing of PHA that provide environmentaladvantages and carbon cost efficiencies (by utilizing carbon-containinggases as a source of carbon for PHA production). Several embodimentsalso reduce one or more of the energy, chemical, and carbon input-to-PHAoutput ratio for producing carbon emissions-based PHA. As such, inseveral embodiments, the systems, methods and materials disclosed hereinrender carbon gas-derived PHA economically competitive withpetrochemical-based plastics.

In some embodiments, the invention also relates to a process thatgenerates a carbon emissions-based PHA material that is cost-competitivewith both food crop-based PHAs and fossil fuel-based thermoplastics.

While PHAs are widely considered to be noncompetitive with fossilfuel-based plastics on energy, chemical, and cost efficiency, severalembodiments of the invention relate to a process for producing PHAs fromcarbon-containing gases that yields unexpectedly improved energy,carbon, chemical, and cost efficiencies over sugar-based PHA productionmethodologies.

Applicant has advantageously discovered that certain culturing orbioprocessing techniques disclosed herein are suitable for production ofPHAs by methanotrophic, heterotrophic, and/or autotrophic microorganismsat efficiencies (e.g., intracellular PHA concentrations, PHA:non-PHAbiomass ratios, or PHA:biocatalyst ratios) that previously were notconsidered achievable. In some embodiments, the techniques relate to themanipulation of the culture or biocatalyst operating environment (e.g.,alteration of one or more of constituents of the culture or nutrientmedia, for example the increase, reduction, or elimination of a certainnutrient (or nutrients), alterations in carbon sources, alterations oftimes or temperatures that certain metabolic activities are underway,and the like).

As a result of such methods, there is provided herein, in severalembodiments, a culture of methanotrophic microorganisms that (a) doesnot express or contain the genetic material encoding soluble methanemonooxygenase, (b) expresses or contains the genetic material encodingthe ethylmalonyl-CoA pathway, and (c) produces polyhydroxyalkanoate(PHA) at intracellular concentrations wherein the ratio of PHA tonon-PHA biomass exceeds about 2:1, about 3:1, about 4:1, about 5:1,about 6:1, about 7:1, about 8:1, about 9:1 (or more) on a dry weightbasis. Additionally, there is provided a composition comprising anisolated microorganism from such a culture.

There is also provided herein, in several embodiments, a culture ofmethanotrophic microorganisms consisting essentially of microorganismsthat (a) do not express or contain the genetic material encoding solublemethane monooxygenase, (b) do express or contain the genetic materialencoding the ethylmalonyl-CoA pathway, and (c) producepolyhydroxyalkanoate (PHA) at intracellular concentrations wherein theratio of PHA to non-PHA biomass exceeds about 3:1, about 4:1, about 5:1,about 6:1, about 7:1, about 8:1, about 9:1 (or more) on a dry weightbasis. Additionally, there is provided a composition comprising anisolated microorganism from such a culture.

There is additionally provided, in several embodiments, a culture ofmethanotrophic microorganisms having (a) particulate methanemonooxygenase activity in the presence of copper ion concentrationsbetween 0.001 micromolar and 1000 micromolar and (b) ethylmalonyl-CoApathway activity in the presence of copper concentrations between 0.001micromolar and 1000 micromolar, wherein said culture is suitable forgeneration of polyhydroxyalkanoate (PHA) at a ratio of PHA to non-PHAbiomass exceeding about 2.9:1, about 3.9:1, about 4.9:1, about 5.9:1,about 6.9:1, about 7.9:1, about 8.9:1 (or more) on a dry weight basis.Additionally, there is provided a composition comprising an isolatedmicroorganism from such a culture.

In several embodiments, the cultures comprise microorganisms of a genusselected from a group consisting of: Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas. In some embodiments,said microorganisms are mutants of microorganisms in the generadisclosed herein. As used herein, the term “mutant” shall be given itsordinary meaning and shall also include genetically-engineered mutants,genetically-manipulated variants, selection-pressure-induced mutants,and the like. Selection-pressure-induced mutation, as used herein, shallbe given its ordinary meaning and shall also refer to conditions inwhich the methanotrophic microorganism is cultured that favor one ormore microorganisms having certain characteristics. Depending on theembodiments, this may be, for example, ability to upregulate or utilizea particular biochemical or metabolic pathway in response to one or morevariables in the culture environment (e.g., nutrients, populationdensity, pH, temperature, salinity, etc.). In several embodiments, theselection-pressure-induced mutation shall also refer to conditions thatexploit certain competitive advantages possessed by certainmethanotrophic microorganisms and not by others (or possessed orexploited to a lesser extent). In additional embodiments, saidmicroorganisms are epigenetically modified, epigenetically engineered,and/or epigenetically mutated (to produce mutants) such that themicroorganisms are capable of producing PHA from carbon-containing gasat high efficiency. As used herein, the terms epigenetic modification,epigenetic mutation, and epigenetic engineering shall be given theirordinary meaning and shall include modification of the expression of thegenetic material comprising one or more microorganism, such that themicroorganism is caused to produce functionally modified materials, suchas enzymes, catalysts, de-coupled feedback loops, conditions-sensitiveenzymes, and polymers. In one embodiment, epigenetic engineering is usedto cause one or more microorganism to produce one or more biocatalyticpathway that is enhanced for high efficiency PHA production, such thatthe microorganism is capable of converting or caused to convertmetabolized carbon into PHA rather than carbon dioxide, protein, orother non-PHA material. In one embodiment, epigenetic modification isused to cause a greenhouse-gas metabolizing microorganism to produce abiocatalytic pathway (e.g., a pathway of enzymes) that is able toproduce PHA at high efficiency. In another embodiment, epigeneticmodification is used to cause a greenhouse-gas metabolizingmicroorganism to produce a biocatalytic pathway (e.g., a pathway ofenzymes) that has a deactivated PHA overproduction control mechanism, orfunctional equivalent thereof, such that the pathway is able to generate(and, in several embodiments, does generate) PHA at high concentrationsrelative to non-polymer material (e.g., about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10 or more units,by weight, of PHA per 1 unit of biomass), wherein such biocatalyticpathway is otherwise not able (in the absence of epigenetic engineering)to produce PHA at concentrations exceeding 0.5, 0.75, 1, 2, or 3 unitsPHA per 1 unit biomass (wherein biomass is, e.g., biocatalyst,non-polymer biomass, and/or enzymes produced by a microorganism). In oneembodiment, epigenetic engineering is used to produce a biocatalyticpathway (wherein the biocatalyst or biocatalytic pathway comprises,e.g., a conglomerate of enzymes and/or associated feedback and/orproduction controls that may be contained inside of a microorganism,expressed on the surface of a microorganism, or produced and used as anextracellular or isolated enzyme or conglomerate of enzymes) containinga production control switch, or the functional equivalent thereof,wherein such switch may be regulated (e.g., turned up or down, on oroff, low or high, and specifically, turned off to cause theoverproduction of PHA relative to non-PHA biomass) according to theconcentrations of various proximate factors, including, but not limitedto nutrient/chemical concentrations (e.g., such as oxygen, carbondioxide, methane, volatile organic compounds, methanol, ethanol,propane, ethane, acetic acid, nitrogen, phosphorus, calcium, magnesium,copper, iron, molybdenum, zinc, aluminum, cobalt, etc.), the relativeratio of nutrient/chemical concentrations, the concentration ofproximate or comixed gases (dissolved or undissolved, gaseous or aqueousform, chemically bound or unbound, physically concentrated or physicallyunconcentrated), shear stress, and/or ultrasonic stress. In oneembodiment, epigenetic modification is caused by modifying theenvironmental conditions impacting the methylation of microorganism DNAor other functional agent impacting the expression or replication ofvarious components, such as gene segments, of microorganism DNA, such asstress conditions, e.g., ultrasonic induction or nutrient over/underaddition, relative nutrient concentrations, which may be uniquely high,low, or unbalanced (e.g., nutrients such as oxygen, nitrogen, carbon,molybdenum, cobalt, iron, copper, phosphorus, magnesium, zinc, andaluminum). In additional embodiments, such DNA expression modificationis used to produce a microorganism or a conglomerate of microorganismsthat contains a genetically-enhanced metabolic pathway, wherein suchpathway is capable of converting carbon into PHA at uniquely highPHA-to-biomass ratios. In another embodiment, such DNA expressionmodification via epigenetic mutation is used to cause microorganisms toproduce enzymes that possess the ability to produce PHA at very highPHA-to-enzyme concentrations (e.g., above about 3:1, about 4:1, about5:1, about 6:1, about 7:1, about 8:1, or greater) and at very high orefficient material synthesis rates, wherein such enzymes have ahypoactive PHA production control mechanism, wherein such enzymes have aPHA production control mechanism that can be turned off, reduced,suppressed, or regulated down, wherein such enzymes can be manipulatedto produce PHA synthase that is not disabled by high PHA concentrations,wherein such enzymes can be rapidly switched from polymerization toprotein production functions.

There are also provided herein, in several embodiments, methods forproducing or mutating a microorganism culture such that the culture canproduce polyhydroxyalkanoate (PHA) at intracellular or extracellularconcentrations exceeding about 60, about 70, about 80, or about 90% byweight, the method comprising: (a) providing a nutrient/culture brothcomprising methane, one or more nutrients comprising copper, oxygen,iron, magnesium, phosphorus, molybdenum, cobalt, sulfur, carbon, zinc,potassium, and/or other functional nutrients impacting microorganismmetabolism, and a methanotrophic, heterotrophic, and/or autotrophicmicroorganism; (b) controlling the concentration of one or morenutrient, such as copper, oxygen, iron, magnesium, phosphorus,molybdenum, cobalt, sulfur, carbon, zinc, potassium, and/or otherfunctional nutrients in said medium to a concentration that can enablethe microorganism(s) to reproduce, and (c) increasing the concentrationof one or more nutrient in said medium to cause said culture orbacterium to produce PHA or increase the production of PHA, including atthe expense of carbon dioxide or protein.

There are also provided herein, in several embodiments, methods forproducing or mutating a microorganism culture such that the culture canproduce polyhydroxyalkanoate (PHA) at intracellular or extracellularconcentrations exceeding about 60, about 70, about 80, or about 90% byweight, the method comprising: (a) providing a nutrient/culture brothcomprising methane, one or more nutrients comprising copper, oxygen,iron, magnesium, phosphorus, molybdenum, cobalt, sulfur, carbon, zinc,potassium, and/or other functional nutrients impacting microorganismmetabolism, and a methanotrophic, heterotrophic, and/or autotrophicmicroorganism; (b) controlling the concentration of one or morenutrient, such as copper, oxygen, iron, magnesium, phosphorus,molybdenum, cobalt, sulfur, carbon, zinc, potassium, and/or otherfunctional nutrients in said medium to a concentration that can enablethe microorganism(s) to produce enzymes, intracellularly orextracellularly, capable of synthesizing PHA, (c) increasing theconcentration of one or more nutrient in said medium to cause saidenzymes to produce PHA or increase the production of PHA relative tocarbon dioxide or protein production, including at the expense of carbondioxide or protein production.

There are also provided herein, in several embodiments, methods forproducing or mutating a microorganism culture such that the culture canproduce polyhydroxyalkanoate (PHA) at intracellular or extracellularconcentrations exceeding about 60, about 70, about 80, or about 90% byweight, the method comprising: (a) providing a nutrient/culture brothcomprising methane, one or more nutrients comprising copper, oxygen,iron, magnesium, phosphorus, molybdenum, cobalt, sulfur, carbon, zinc,potassium, and/or other functional nutrients impacting microorganismmetabolism, and a methanotrophic, heterotrophic, and/or autotrophicmicroorganism; (b) controlling the concentration of one or morenutrient, such as copper, oxygen, iron, magnesium, phosphorus,molybdenum, cobalt, sulfur, carbon, zinc, potassium, and/or otherfunctional nutrients in said medium to a concentration that can enablethe microorganism(s) to intracellularly or extracellularly produceenzymes capable of synthesizing PHA, (c) separating the enzymes from themicroorganism, (d) contacting carbon with the enzymes, and (e)increasing the concentration of one or more nutrient in said medium tocause said enzymes to produce PHA or increase the production of PHArelative to carbon dioxide or protein production, including at theexpense of carbon dioxide or protein production.

There are also provided herein, in several embodiments, methods forproducing or mutating a methanotrophic culture or bacterium such thatthe culture or bacterium can produce polyhydroxyalkanoate (PHA) atintracellular concentrations exceeding about 60, about 70, about 71,about 80, or about 90% by weight in a non-sterile environment, themethod comprising: (a) providing a culture broth comprising methane, amedium comprising one or more nutrients comprising copper, and amethanotrophic culture or bacterium; (b) controlling the concentrationof copper in said medium to a concentration that can enablemethanotrophic microorganisms to produce sMMO, (c) reducing theconcentration of one or more nutrient in said medium to cause saidculture or bacterium to produce PHA, (d) increasing the concentration ofsaid one or more nutrient of step (c) to cause said culture or bacteriumto reproduce using essentially only pMMO, and (e) subjecting saidculture or bacterium to at least two repetitions of steps (b), (c), and(d).

In several embodiments, there is provided PHA produced by the culturesor bacterium disclosed herein. In some embodiments, there is providedPHA comprising a culture or bacterium as disclosed herein.

In several embodiments, there is provided the use of microorganismshaving the characteristics of (a) producing no soluble methanemonooxygenase (or no detectable soluble methane monooxygenase) at anycopper concentration, and/or not expressing or containing the geneticmaterial encoding soluble methane monooxygenase; (b) expressing theethylmalonyl-CoA metabolic pathway; and (c) the capability of producingpolyhydroxyalkanoate at intracellular concentrations exceeding about 72%by dry weight, wherein said microorganisms are selected from the groupof genera consisting of Methylosinus, Methylocystis, Methylococcus,Methylobacterium, Pseudomonas, and mutants thereof, to producepolyhydroxyalkanoate.

In several embodiments, there is also provided the use of microorganismshaving the characteristics: (a) producing no soluble methanemonooxygenase (or no detectable soluble methane monooxygenase) at anycopper concentration (or do not express or contain the genetic materialencoding soluble methane monooxygenase); (b) express theethylmalonyl-CoA metabolic pathway; and (c) the capability to producepolyhydroxyalkanoate at intracellular concentrations exceeding about 57%by dry weight; wherein said microorganisms are selected from the groupof genera consisting of Methylosinus, Methylocystis, Methylococcus,Methylobacterium, Pseudomonas, and mutants thereof, to producepolyhydroxyalkanoate

There is also provided the use of microorganisms having thecharacteristics (a) produce no soluble methane monooxygenase (or nodetectable soluble methane monooxygenase) at any copper concentration(or do not express or contain the genetic material encoding solublemethane monooxygenase); (b) express the ethylmalonyl-CoA metabolicpathway; and (c) capable of producing polyhydroxyalkanoate atintracellular concentrations exceeding about 23% by dry weight; whereinsaid microorganisms are selected from the a group consisting ofMethylosinus, Methylocystis, Methylococcus, Methylobacterium,Pseudomonas, and mutants thereof, to produce polyhydroxyalkanoate.

There is provided, in several embodiments, a method for enhancingpolyhydroxyalkanoate (PHA) generation in a methanotrophic culture byreducing copper concentrations to effect particulate methanemonooxygenase (pMMO) production comprising contacting a culture ofmethanotrophic microorganisms with a medium comprising copper, one ormore additional nutrients, and a carbon-containing gas that can bemetabolized by the culture, incubating the culture in the medium tocause growth of the culture, inducing a selection pressure in theculture to transform the culture into a culture that generates PHApreferentially through pMMO by: (i) reducing the concentration of copperin the medium to cause production of soluble methane monooxygenase(sMMO) and/or particulate methane monooxygenase by the culture, whereinthe concentration of copper causes the production of sMMO in somemethanotrophic microorganisms; (ii) reducing the concentration of one ormore the nutrient in the medium to cause the culture to generate PHAfrom the carbon-containing gas using the sMMO or the pMMO, wherein PHAis generated by pMMO at a greater rate as compared to sMMO, (iii)returning the culture to the growth conditions, wherein microorganismshaving higher intracellular concentrations of pMMO and PHA grow at agreater rate as compared to those with lower intracellular pMMO and PHAconcentrations; and (iv) repeating steps (ii) and (iii), wherein therepetitions selectively favor growth of microorganisms that produce PHAvia pMMO, thereby facilitating the pMMO-mediated production of PHA atreduced copper concentrations, and resulting in a culture comprisingessentially only microorganisms that use pMMO to produce PHA.

There is also provided a method for enhancing particulate methanemonooxygenase (pMMO) mediated polyhydroxyalkanoate (PHA) generationcomprising contacting a culture of methanotrophic microorganisms with amedium comprising copper, nitrogen, one or more additional nutrients,and a carbon-containing gas that can be metabolized by the culture,incubating the culture in the medium to cause growth of the culture; andtransforming the culture into a culture that generates PHApreferentially through pMMO by: (i) reducing the concentration of copperin the medium to cause production of soluble methane monooxygenase(sMMO) and particulate methane monooxygenase by the culture, wherein theconcentration of copper favors the production of sMMO; (ii) reducing theconcentration of nitrogen to cause the culture to generate PHA from thecarbon-containing gas using the sMMO or the pMMO, wherein PHA isgenerated by pMMO at a more rapid rate as compared to sMMO, resulting ina portion of the microorganisms having higher intracellularconcentrations of PHA as compared to those using sMMO; and (iii)returning the culture to the growth conditions, wherein themicroorganisms having higher intracellular concentrations of PHA grow ata greater rate as compared to those with lower intracellular PHAconcentrations; and (iv) repeating steps (ii) and (iii), wherein therepetitions selectively favor growth of microorganisms that produce PHAvia pMMO, thereby facilitating the pMMO-mediated production of PHA.

There is also provided, in several embodiments, a method for convertinga carbon-containing material into a polyhydroxyalkanoate (PHA), themethod comprising (a) contacting a culture of methanotrophicmicroorganisms with a medium comprising copper, one or more additionalnutrients, and a carbon-containing material that can be metabolized bythe culture, thereby inducing growth of the culture (b) reducing orcontrolling the concentration of copper in the medium to cause at leasta portion of the culture to produce soluble methane monooxygenase (sMMO)and/or particulate methane monooxygenase (pMMO), wherein the copperconcentration can cause one or more methanotrophic microorganism toproduce sMMO, (c) reducing the concentration of at least one of thenutrients in the medium to cause at least a portion of the culture toutilize the produced sMMO or the produced pMMO to convert the carbonfrom the carbon-containing material into PHA, (d) repeating steps (a)through (c) a plurality of times, wherein the portion of the cultureutilizing the pMMO to generate PHA grows and/or generates PHA at agreater rate than the portion of the culture utilizing the sMMO togenerate PHA, thereby inducing a selective pressure in the cultureresulting in a culture comprising essentially only microorganisms thatuse pMMO to produce PHA.

In several embodiments, the induced selection pressure selects formicroorganisms in which the presence and/or expression of the geneencoding the sMMO is reduced. This is unexpected, in severalembodiments, as the copper concentrations are such that sMMO productionwould typically be favored. When employed in combination with periods ofgrowth, PHA production, and growth, the selection pressure unexpectedlyand advantageously allows those microorganisms operating through thepMMO pathway to become grow more quickly, thereby outcompeting many, ifnot all, organisms using an alternate MMO pathway. In severalembodiments, as the growth-polymerization-growth cycles are repeated inconjunction with the existence of conditions favoring pMMO metabolism,the expression of the gene encoding the soluble methane monooxygenaseenzyme is reduced. Thus, the growth advantage of those microorganismsutilizing pMMO provides, in each cycle, at least a marginal gain withrespect to the sMMO-utilizing microorganisms increasing their percentagecontribution to the overall demographics of the culture. With eachsuccessive round, pMMO-utilizing microorganisms outcompete thesMMO-utilizing microorganisms, thereby eventually being the primary, ifnot only, microorganism in the culture. As a result this process, the“weaker” sMMO-utilizing microorganisms will be reduced in number, and insome cases may undergo an evolutionary-pressure induced change toreduce/eliminate sMMO expression in favor of pMMO.

In additional embodiments, alternative methods may be used to achieve areduction in the presence and/or expression of the sMMO gene and/orprotein. For example, as disclosed herein genetically modified (alsoreferred to herein as genetically-engineered) microorganisms may be usedin certain embodiments. Thus, prior to a culturing process in which PHAwould be produced, in some embodiments, microbiological techniques areused to excise the genetic material encoding sMMO from the genome of themicroorganism. Similarly, microorganisms having the genetic material forsMMO could be cultured with (e.g., bred with) an alternative strain ofmicroorganisms having genetic material for a metabolically favorable(e.g., more active) pMMO. After successive rounds of crossbreeding,microorganisms are selected based on a combination of limited sMMOexpression and robust high activity pMMO expression. In additionalembodiments, methods can be used to suppress one or more of theexpression where the activity of the sMMO enzyme, rather thanmanipulating the microorganism on genomic level. For example, RNAinterference or antisense RNA could be used to suppress expressionand/or function of sMMO. Similarly site directed mutagenesis could beused such that a microorganism would produce a mutant (e.g.,nonfunctional or minimally functional) sMMO. Site directed mutagenesiscan also be employed to introduce a specific mutation in the sMMO genesuch that a truncated and/or nonfunctional (or reduced function) S MMOis produced. For example, the introduction of mutation that results inearly placement of a stop codon in the sMMO gene would result in atruncated, and likely nonfunctional enzyme (if any enzyme and all werein fact be produced). In addition to, or in place of such approaches,inducible or repressible promoters could be employed such that sMMOwould only be expressed under certain conditions (e.g., the addition ofan inducing agent to the culture media) or alternatively would normallybe repressed absent the removal of repressing agent from the media. Akinto gene therapy approaches, microorganisms could be modified to carryexogenous DNA that produces a repressor of sMMO function (oralternatively, a promoter of pMMO function).

In some embodiments, the actual and/or bioavailable concentration of thecopper in the medium is reduced to a concentration less than about 0.001mg/L, less than about 0.5 micromolar, less than about 0.1 mg per dryweight gram of the microorganisms, less than about 100 mg/L, or lessthan about 1 mg/L. In some embodiments, the concentration of copper ismaintained at the reduced level. In some embodiments, the copperconcentration is returned to initial concentrations. In severalembodiments, reduction of one or more additional nutrient comprises asubstantial depletion of nitrogen from the media. Substantial depletioncomprises, in some embodiments, depletion of nitrogen concentrations byabout 50%, about 60%, about 70%, about 80%, about 90%, about 95%, ormore. In other embodiments, depending on the metabolic status of theculture, smaller reductions in nitrogen, another nutrient in the media,or combinations thereof may be used to effect PHA polymerization.

In several embodiments, the carbon-containing gas that the culture usesas a source of carbon to generate PHA comprises methane. In someembodiments, the carbon-containing gas comprises carbon dioxide. In someembodiments, the carbon-containing gas comprises one or more volatileorganic compounds. In some embodiments, one or more of methane, carbondioxide, volatile organic compounds, and other gasses or compounds maybe present in the carbon-containing gas.

In some embodiments, the carbon source need not be a gas. Acarbon-containing material comprising a non-gaseous material (eitheralone or in combination with a gas) may also be used in certainembodiments. For example, in some embodiments, the non-gaseous materialcomprises one or more volatile fatty acids, methanol, acetate, acetoneor acetic acid, formate, formaldehyde or formic acid, propane, ethane,or combinations thereof (or in combination with other metabolizablecarbonaceous materials). In addition, in some embodiments, thenon-gaseous material comprises microorganism biomass removed from onepolymerization stage and added to the medium in a subsequent growthstage. In one embodiment, methanotrophic microorganisms are exposed toacetic acid and/or acetate to cause said microorganisms to reproduce orgenerate chemicals, such as PHA. In another embodiment, methanotrophicmicroorganisms are exposed to acetic acid and/or acetate together withmethane, independent of methane, or periodically with methane andperiodically without methane, wherein said acetate or acetic acid may bederived by chemically, electrochemically, and/or biologically convertingcarbon dioxide into acetic acid or acetate, wherein non-obligate(facultative) methanotrophic microorganisms use such acetic acid oracetate to produce biomass, protein, PHA, methanol, butanol, cartenoids,chemicals, or other materials. In one embodiment, electrical current isadded to a methanotrophic culture to cause the culture to produce higheramounts of PHA than the culture would produce without the addition of anelectrical current. In another embodiment, methanotrophic microorganismsare exposed to concentrations of nitrogen, potassium, magnesium, carbondioxide, methane, dinitrogen, or other chemicals, and particularlycarbon dioxide, that induce said microorganisms to produce one or morechemicals, such as methanol, butanol, ethanol, or other alcohols, andparticularly methanol, wherein said alcohol is excreted extracellularly,wherein the concentration of gases/nutrients, such as carbon dioxide,nitrogen, potassium, magnesium, methane, dinitrogen, or other chemicals,is used to induce, control (e.g., maintain), and/or increase theextracellular production of alcohol or other chemicals from saidmicroorganisms. In another embodiment, a culture of methanotrophic,heterotrophic, and/or autotrophic microorganisms are contacted with anelectric current or other agent, wherein carbon dioxide is converted toacetic acid or acetate, wherein such acetic acid or acetate issubsequently utilized as a source of carbon by said microorganisms toproduce PHA, chemicals, proteins, or other materials, wherein suchcarbon dioxide may or may not be derived as a metabolic byproduct ofmethanotrophic metabolism. In another embodiment, a method is providedfor converting carbon dioxide into PHA or other materials using amethanotrophic microorganism, the method comprising converting carbondioxide into acetic acid or acetate and inducing a methanotrophicmicroorganism to convert such acetic acid or acetate into biomass,protein, PHA, methanol, butanol, or other materials, wherein suchprocess of converting carbon dioxide to acetic acid or acetate (or othercarbon-containing chemical that can be assimilated and metabolized byeither a methanotrophic microorganism or other microorganism, such as aheterotrophic microorganism) and subsequently converting such aceticacid, acetate, or carbon-containing chemical into PHA, biomass,methanol, butanol, or another chemical takes place in one, two, ormultiple vessel(s). In one embodiment, methanotrophic microorganisms arecaused to increase the production of extracellularly excreted methanol,butanol, ethanol, other liquids or volatile organic compounds, lipids,PHAs, or proteins, which can be excreted in media or gas, by increasingthe actual or relative concentration of a nutrient, wherein suchnutrient may be one or more of carbon, hydrogen ions, hydroxide ions,oxygen, hydrogen, carbon dioxide, methane, nitrogen, dinitrogen, urea,acetic acid, volatile organic compounds, organic compounds, inorganiccompounds, silicon, magnesium, sodium, EDTA, calcium, phosphorus, zinc,cobalt, cadmium, aluminum, potassium, electrons, or other nutrient ormetabolism-influencing material, and particularly carbon dioxide,magnesium, electrons, and/or sodium, thereby, in one embodiment,inducing a metabolic shift in the proclivity or ability of amethanotroph to produce or use methanol-metabolizing enzymes. In oneembodiment, such extracellularly excreted chemical (such as methanol,ethanol, or PHA) is harvested by heating the liquid media, subjectingthe liquid media or proximate gas to a scrubber, such as a waterscrubber to absorb, e.g., alcohols, subjecting liquid media to a vacuumto cause such chemical to vaporize, subjecting the media to apervaporator, and/or subsequently reducing the concentration of water inthe chemical, or reducing the concentration of chemical in the water, orreducing the concentration of chemical in the proximate gas stream. Inone embodiment, the production of proteins, PHAs, or other chemicals,such as diesel, methanol, gasoline, butanol, vaccines, therapeutics, andother chemicals, may be carried out using methanotrophic microorganismsas a method to avoid one or more Maillard reactions. Sincemethanotrophic metabolism neither requires nor inherently producessugars, methanotrophy, in one embodiment, is used as a platform to avoidMaillard reactions in the production of proteins and other chemicals. Inone embodiment, methanotrophic microorganisms are used to producematerials that are currently challenged by the occurrence (and resultinghindrance) of one or more Maillard reactions, such as in the productionof antibiotics, antibodies, pharmaceuticals, and/or platform chemicals.In one embodiment, methanotrophic microorganisms are employed to producematerials in processes while avoiding Maillard reactions or the productsof glycation.

Advantageously, the methods disclosed herein allow production PHA athigh intracellular concentrations. The concentrations, in someembodiments, are at least 70% of the dry biomass weight of themicroorganisms. In some embodiments, the concentrations are particularlyadvantageous because the high intracellular PHA concentration enablesmore rapid growth of those microorganisms. Thus, a feed-forward cycleexists, in some embodiments, in that a microorganism that is selectedfor by the induced pressures produces PHA more rapidly through the pMMOpathway, thereby having a greater concentration of PHA, which furtherimparts the ability to grow more rapidly (vis-à-vis those microorganismswith lower PHA concentrations, such as those utilizing the sMMOpathway). As the process is repeated, the pMMO-utilizing microorganismspossess a growth advantage at (at least) two stages in the process,thereby allowing their dominance in the culture. As such, in severalembodiments, the culture comprising essentially only microorganisms thatuse pMMO to produce PHA comprises a culture wherein over about 50% ofthe culture uses pMMO to produce PHA. Additionally, in severalembodiments, the culture comprising essentially only microorganisms thatuse pMMO to produce PHA comprises over 80% of the culture. In severalembodiments, the microorganisms in the culture are from the genusMethylocystis.

In addition, several embodiments, the repeated cycling between growth,copper reduction to favor pMMO production, PHA production and back togrowth also favors the production of microorganisms that possess thegenetic material encoding the ethylmalonyl-CoA pathway. Advantageously,this pathway allows, in some embodiments, additional carbon sources tobe used by the culture, thereby broadening the range of substrates thatcan eventually be used as substrates for the generation of PHA.

A method for converting a carbon-containing material into apolyhydroxyalkanoate is also provided in several embodiments, the methodcomprising: (a) contacting a methanotrophic culture with a mediumcomprising one or more nutrients and a carbon-containing material thatcan be metabolized by the culture, (b) controlling the concentration ofthe one or more nutrients in the medium to cause at least a portion ofthe culture to produce sMMO and/or pMMO, (c) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA; (d) repeating steps (a) through (c) a pluralityof times.

In several embodiments, the nutrient of step (b) and the nutrient ofstep (c) are the same nutrient, while in other embodiments, the nutrientof step (b) and the nutrient of step (c) are different nutrients. In oneembodiment, the nutrient is selected from the group consisting ofcopper, methane, oxygen, phosphorus, potassium, magnesium, boron,sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof.

Also provided for in several embodiments herein is a method forconverting a carbon-containing material into a polyhydroxyalkanoate(PHA), the method comprising: (a) providing a microorganism culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture, wherein the genetic material encoding the ethylmalonyl-CoApathway is present in the one or more microorganisms, (d) controllingthe concentration of the one or more nutrient in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d).

In several embodiments, the nutrient is copper, and step (c) comprisesperiodically or permanently reducing the concentration of the copper inthe medium. In several embodiments, the actual and/or bioavailableconcentration of the copper in the medium to be less than about 0.001mg/L, less than about 0.5 micromolar, less than about 0.1 mg per dryweight gram of the microorganisms, less than about 100 mg/L, or lessthan about 1 mg/L. In several embodiments, controlling the actual and/orbioavailable concentration of the copper in the medium effects theproduction of microorganisms that do not possess the gene encodingsoluble methane monooxygenase or express sMMO at reduced amounts. Inother embodiments, the organisms have the gene, but the enzyme is notexpressed.

In some embodiments, the nutrient is selected from the group consistingof copper, methane, oxygen, phosphorus, potassium, magnesium, boron,sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof, and step (c) comprises periodically or permanently increasing,reducing, or maintaining the concentration of the nutrient in the mediumto effect the production of microorganisms that do not possess the geneencoding soluble methane monooxygenase. In several embodiments, thenutrient of step (c) and the nutrient of step (d) are the same nutrient,while in other embodiments, the of nutrient of step (c) and the nutrientof step (d) are different nutrients.

In several embodiments, steps (a) through (d) are repeated a pluralityof times and the concentration of the one or more microorganisms in theculture increases as the steps (a) through (d) are repeated.

There is also provided, in several embodiments, a method for convertinga carbon-containing material into a polyhydroxyalkanoate (PHA), themethod comprising: (a) providing a microorganism culture, (b) providinga medium comprising one or more nutrient comprising a carbon-containingmaterial that can be metabolized by the culture, (c) controlling theconcentration of the one or more nutrient in the medium to cause thecellular replication of one or more microorganisms in the culture,wherein the genetic material encoding the ethylmalonyl-CoA pathway ispresent in the one or more microorganisms, and wherein the expression ofsoluble methane monooxygenase is reduced in the one or moremicroorganisms as compared to a first instance of step (a), (d)controlling the concentration of the one or more nutrient in the mediumto cause the culture to produce PHA, and (e) repeating steps (a) through(d).

Copper concentrations can be reduced, in several embodiments, asdiscussed herein, e.g., to concentrations less than about 0.001 mg/L,less than about 0.5 micromolar, less than about 0.1 mg per dry weightgram of the microorganisms, less than about 100 mg/L, or less than about1 mg/L. Also as discussed above, the nutrients can be the same invarious steps, or alternatively they may be different. In oneembodiment, for example, the nutrient of step (c) is selected from thegroup consisting of methane, oxygen, phosphorus, potassium, magnesium,boron, sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof and the nutrient of step (d) is selected from the groupconsisting of methane, oxygen, phosphorus, potassium, magnesium, boron,sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof, but is not the same as the nutrient of step (c).

There is also provided a method for controlling the functionalcharacteristics of polyhydroxyalkanoate (PHA) produced by amethanotrophic culture exposed to methane and one or more non-methanematerials that influence the metabolism of the culture, the methodcomprising: (a) providing a methanotrophic culture in a mediumcomprising one or more nutrients, (b) controlling the concentration ofone or more of the nutrients in the medium to cause the culture toproduce soluble methane monooxygenase (sMMO) and/or particulate methanemonooxygenase (pMMO), (c) controlling the concentration of the one ormore nutrients in the medium to cause the culture to produce PHA, and(d) repeating steps (a) through (c), wherein the concentration of sMMOrelative to pMMO in step (c) is substantially the same in eachrepetition, wherein one or more functional characteristics of the PHAproduced by the culture are controlled in multiple PHA productionrepetitions, and wherein the culture selectively generatesmicroorganisms that synthesize PHA at high efficiency.

In several embodiments, the concentration of the sMMO relative to thepMMO in the culture is substantially the same in step (c) in at leasttwo consecutive repetitions. Advantageously, this consistency results ina more defined and predictable PHA. For example, in several embodiments,PHA produced by the culture exhibits substantially the same of one ormore of the following characteristics in one or more the repetitions:molecular weight, polydispersity, impact strength, elasticity,elongation, and/or modulus. As discussed above, a variety of carbonsources may be used. In some embodiments, non-methane sources are used,such as carbon dioxide, volatile organic compounds, volatile fattyacids, methanol, acetate, acetone, and acetic acid, formate,formaldehyde, and formic acid, propane, ethane, oxygen, acarbon-containing material that can be metabolized by the culture, orcombinations thereof.

In one embodiment, at least about 60% of microorganisms in the cultureproduce only the pMMO, while in one embodiment at least about 60% ofmicroorganisms in the culture produce only the sMMO. In still additionalembodiments, the culture comprises an equal concentration of the sMMOand the pMMO. In one embodiment, the concentration of the sMMO is morethan 2 times greater than the concentration of the pMMO in the culture.In one embodiment, the concentration of the sMMO is more than 5 timesgreater than the concentration of the pMMO in the culture. In oneembodiment, the concentration of the sMMO is more than 10 times greaterthan the concentration of the pMMO in the culture. In other embodiments,the concentration of the pMMO is more than 2 times greater than theconcentration of the sMMO in the culture. In one such embodiment, theconcentration of the pMMO is more than 5 times greater than theconcentration of the sMMO in the culture.

There is also provided a method for modifying the functionalcharacteristics of a polyhydroxyalkanoate (PHA) material, comprising thesteps of: (a) providing a PHA and a biomass, (b) subjecting the PHAand/or the biomass to a processing step in order to render the at leasta portion of the biomass miscible with the PHA, (c) combining the PHAand the biomass in a mixture to form a compound, (d) heating thecompound to between 50 degrees Celsius and 250 degrees Celsius, and (e)causing the biomass to effect a functional modification of the PHA,wherein the functional modification comprises plasticization,nucleation, compatibilization, melt flow modification, strengthening,reduction of PHA crystallinity or rate of crystallization, increase inoptical clarity, and/or elasticization. In one embodiment, there is alsoprovided a method for modifying the functional characteristics of apolyhydroxyalkanoate (PHA) material, comprising the steps of: (a)providing a PHA and a biomass, (b) subjecting the PHA and/or the biomassto a processing and modification step wherein the PHA and the biomassare subject to temperatures between at least 20 degrees Celsius and 250degrees Celsius and to pressures of at least between 1 atmosphere and350 atmospheres, thereby causing the modified biomass to effect afunctional modification of the PHA material, wherein the functionalmodification comprises an increase in plasticization, nucleation,compatibilization, melt flow modification, strengthening, and/orelasticization. In one embodiment, the ratio of biomass to PHA in saidPHA material may be between about 1:1000 and about 1000:1, includingbetween about 1:1000 and about 1:500, about 1:500 and about 1:100, about1:100 and about 1:10, about 1:10 and about 1:9, about 1:9 and about 1:8,about 1:8 and about 1:5, about 1:5 and about 1:3, about 1:3 and about1:1, about 1:1 and about 2:1, about 2:1 and about 5:1, about 5:1 andabout 10:1, about 10:1 and about 100:1, about 100:1 and about 500:1, andabout 500:1 about 1000:1, and overlapping ratios therein. In oneembodiment, the temperature, in degree Celsius, of the modification stepmay range from about 20 to about 40, about 40 to about 60, about 60 toabout 80, about 80 to about 100, about 100 to about 120, about 120 toabout 140, about 140 to about 200, about 140 to about 160, about 160 toabout 180, about 180 to about 200, about 200 to about 220, about 220 toabout 250, about 250 to about 300, about 90 to 200, about 140 to about220, about −50 to about 400, and overlapping ranges thereof. In oneembodiment, the pressure, pounds per square inch, of the modificationstep may range from about −30 to about 50,000, about −30 to about 0,about 0 to about 50, about 0 to about 3,000, about 0 to about 200, about0 to about 10,000, about 0 to about 5,000, about 0 to about 20,000,about 20,000 to about 50,000, about 10,000 to about 40,000, about 25,000to about 30,000, about 0 to about 500, about 0 to about 1000, about 0 toabout 2,000, about 40,000 to about 50,000, and overlapping rangesthereof. In one embodiment, the modification step may be used toeliminate the need for one or more of additional plasticization,nucleation, compatibilization, melt flow modification, strengthening,reduction of PHA crystallinity or rate of crystallization, increase inoptical clarity, and/or elasticization.

Advantageously, a variety of types of biomass may be used, for example,one or more of methanotrophic, autotrophic, and heterotrophic biomassare used in several embodiments. In one embodiment, the processingcomprises treating the PHA and/or the biomass with one or more of thetreatments selected from the group consisting of: heat, shear, pressure,solvent extraction, washing, filtration, centrifugation, sonication,enzymatic treatment, super critical material treatment, cellulardissolution, flocculation, acid and/or base treatment, drying, lysing,and chemical treatment.

The biomass may be present at varied concentrations, depending on theembodiment. For example in one embodiment, the biomass is present in thecompound at a concentration of more than 0.001%, while in otherembodiments, the biomass is present in the mixture at a concentration ofmore than 0.01%. In still other embodiments, depending on the make-up ofthe biomass and the characteristics of the PHA, greater or lesserconcentrations may also be used.

The functional characteristics of PHA may also be modified through themelting and cooling of the PHA polymer in the presence of adual-miscible biomass agent and a second polymer by a method provided inseveral embodiments herein, the method comprising, comprising the stepsof: (a) providing a first polymer, a biomass, and a second polymer,wherein the first polymer is a PHA, (b) subjecting the biomass to aprocessing step comprising heat, pressure, solvent washing, filtration,centrifugation, super critical solvent extraction, and/or shear, whereinthe processing step renders at least a portion of the biomass misciblewith the first polymer and the second polymer, (c) contacting the firstpolymer with the biomass and the second polymer to form a compound, (d)heating the compound to between about 50 degrees Celsius and about 250degrees Celsius and adding pressure to the compound between about 0 andabout 50,000 pounds per square inch, thereby causing the biomass toeffect a functional modification of the first polymer, the secondpolymer, and the combination of the first polymer and the secondpolymer, wherein the functional modification comprises plasticization,nucleation, compatibilization, melt flow modification, strengthening,reduction of PHA crystallinity or rate of crystallization, increase inoptical clarity, and/or elasticization. In one embodiment, PHA may bemixed and/or co-melted with a miscible agent to reduce thecrystallinity, increase the clarity, increase the flexibility, and/orlower the melting point of the PHA, wherein such PHA may be PHB, PHBV,PHHX, PHO, or other PHA polymer. In one embodiment, such miscible agentsinclude plasticizers that reduce crystallinity, increase clarity,increase flexibility, and reduce the melt temperature of PHA, inconcentrations ranging from about 0 to about 100%, about 0 to about 30%,about 0 to about 20%, about 0 to about 15%, about 0 to about 10%, about5 to about 10%, about 0 to about 5%, or other concentration. In oneembodiment, the degradation temperature of PHB or PHBV may be increasedby purifying (at least partially) the PHB or PHBV, wherein purifying thePHB or PHBV comprises reducing the concentration of proximate salts,biomass, or other non-PHA materials, thereby reducing the loss of PHAmolecular weight induced by the presence of such non-PHA materials in ahigh temperature condition. In another embodiment, PHA may be degradedinto low molecular weight PHA, such as low molecular weight (MW) PHB, toproduce oligomers, dendrimers, or other low MW derivatives, such thatsuch low MW PHA can be used as a plasticizer, compatibilizer, orfunctional modification agent in PHA, PVC, polyproylene, polyethylene,ABS, TPU, polystyrene, or other chemicals or polymers; such low MW PHAprovides surprisingly useful features as a plasticizer, compatibilizer,and/or as a miscible agent, including compatibilization and/orplasticization with non-PHA materials, such as polyolefins. In anotherembodiment, PHA may be degraded into low molecular weight PHA, crotonicacid, PHA monomers (e.g., such as hydroxybutyrate), or other PHAderivative by thermal degradation, enzymatic degradation, or otherwise,and such low MW PHA or PHA derivatives may be reconstructed as buildingblocks into a polymer or other chemical through the use of a catalyst,compatibilizer, cross-linker, nucleating point, star-block creatingpolymer, random co-polymer, chemical intermediary, or other chemicalagent that serves to use PHA, low MW PHA, or PHA derivatives as unitsfor polymerization or chemical construction. In one embodiment, miscibleagents, such as chemicals with acetic acid or acetyl or acetic groups,are added to PHA to reduce crystallinity and/or increase clarity,wherein a PHA is rendered more amorphous. In other embodiments, theclarity of PHA itself, or the mixture of PHA with other non-PHApolymers, such as polyolefins, PLA, or other polymers, is increased byadjusting the refractive index of either PHA or the non-PHA polymers,such that the refractive indices of the various polymers are caused tocome into closer proximity. In other embodiments, the crystal sizes anddomains of PHA are reduced through the addition of one or morenucleating agents, such as boron nitride (including various grades ofboron nitride, including from various manufacturers providing varyingdegrees of clarity, including from about 0 to about 1%, about 0 to about0.2%, about 0 to about 3%, about 0 to about 0.1%, about 0 to about 10%,about 0 to about 20%, about 1 to about 2%, and various overlappingranges therein); suitable nucleating agents may also include miscibleagents or plasticizers that are caused to dissolve into PHA or otherwiseproduce a homogenous mixture, including nucleating agents that come outof solution below the melt temperature of PHA but form a soluble mixturewith PHA at or above the melting temperature of PHA, thereby forminghighly dispersed, fine, or otherwise effective nucleation points, whichmay reduce crystallinity, reduce haze, increase compatibilization withnon-PHA materials, increase clarity, reduce brittleness, improvetoughness, increase crystallization temperature, and/or reduce secondarycrystallization. In one embodiment, secondary crystallization in a PHAis reduced or eliminated by increasing the PHA crystallizationtemperature by reducing or eliminating the crystallinity of PHA throughthe use, e.g., of plasticization, comixing PHA with other miscibleagents, comixing PHA with polyolefins, adding antioxidants to PHA, oradding compatibilization agents to PHA. In one embodiment, PHAs arecomixed and/or melt blended with other non-PHA polymers or materials,such as polypropylene (homopolymer, copolymer, random copolymer, whereinrandom copolymers are particularly useful by increasing the ethylenecontent of the material to lower melt temperature, increaseco-compatibilization, reduce crystallinity, and increase toughness),glass fibers, organic fibers, inorganic fibers, wood fibers, calciumcarbonate (at various particle size distributions and at variousinjection points, such that CaCO₃ at low/fine particle size will be morefunctional as part of a PHA blend than higher particle size CaCO₃),polyethylene (including linear low density polyethylene, low density PE,high density PE, wherein PE reduces the crystallinity of PHA, includingPHB, reduces the melt temperature, and reduces secondarycrystallization), ABS, glass-filled PP, TPU (including with TPU-PHAcompatibilizers), polystyrene (including high impact polystyrene), PET,PMMA, biomass, wood, hemp fibers, polyolefins, other non-PHA polymers,and/or antioxidants. In some embodiments, PHAs are functionally modifiedand/or enhanced through the addition/compounding with: 1) plasticizers,such as tributyl citrate, at a variety of concentrations, 2) nucleatingagents, such as boron nitride, 3) antioxidants, 4) fibers, 5)cross-linking agents (such as peroxides), 6) low or high glasstransition temperature materials that may be miscible with PHA, 7) lowMW PHAs, such as low MW PHB or PHBV, 8) biomass, and/or biomassderivatives, and/or 8) non-PHA polymers or non-PHA materials. In oneembodiment, the non-PHA polymer or material consists of one or more ofthe following solvents, cell dissolution agents, cell metabolizingagents, polymers, plasticizers, compatibilization agents, miscibleagents, and nucleating agents: polypropylene, polyethylene, polystyrene,polycarbonate, acrylonitrile butadiene styrene, polyethyleneterephthalate, polyvinyl chloride, fluoropolymers, liquid crystalpolymers, acrylic, polyamide/imide, polyarylate, acetal, polyetherimide,polyetherketone, nylon, polyphenylene sulfide, polysulfone, cellulosics,polyester, polyurethane, polyphenylene oxide, polyphenylene ether,styrene acrylonitrile, styrene maleic anhydride, thermoplasticelastomer, ultra high molecular weight polyethylene, epoxy, melaminemolding compound, phenolic, unsaturated polyester, polyurethaneisocyanates, urea molding compound, vinyl ester, polyetheretherketone,polyoxymethylene plastic, polyphenylene sulfide, polyetherketone,polysulphone, polybutylene terephthalate, polyacrylic acid, cross-linkedpolyethylene, polyimide, ethylene vinyl acetate, polyvinyl chloride,polyvinyl acetate, polyvinyl acetate co-polyvinylpyrrolidone,polyvinylpyrrolidone, polyvinyl alcohol, cellulose, lignin, celluloseacetate butryate, polypropylene, polypropylene carbonate, propylenecarbonate, polyethylene, ethyl alcohol, ethylene glycol, ethylenecarbonate, glycerol, polyethylene glycol, pentaerythritol, polyadipate,dioctyl adipate, triacetyl glycerol, triacetyl glycerol-co-polyadipate,tributyrin, triacetin, chitosan, polyglycidyl methacrylate, polyglycidylmethacrylate, oxypropylated glycerine, polyethylene oxide, lauric acid,trilaurin, citrate esters, triethyl citrate, tributyl citrate, acetyltri-n-hexyl citrate, saccharin, boron nitride, thymine, melamine,ammonium chloride, talc, lanthanum oxide, terbium oxide, cyclodextrin,organophosphorus compounds, sorbitol, sorbitol acetal, sodium benzoate,clay, calcium carbonate, sodium chloride, titanium dioxide, metalphosphate, glycerol monostearate, glycerol tristearate,1,2-hydroxystearate, cellulose acetate propionate, polyepichlorohydrin,polyvinylphenol, polymethyl methacrylate, polyvinylidene fluoride,polymethyl acrylate, polyepichlorohydrin-co-ethylene oxide, polyvinylidene chloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHB V, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, styrene maleic anhydride,styrene-acrylonitrile, poly(methyl methacrylate),polytetrafluoroethylene, polybutylene. polylactic acid, polyvinylidenechloride, and/or other similar materials or combinations of thesematerials, including mold release agents, plasticizers, solvents,solvent-grafted polymer, salts, nucleating agents, cross-linking agents,filaments, water, antioxidants, compatibilizers, co-polymers, peroxides,alcohols, ketones, polyolefins, chlorinated solvents, non-chlorinatedsolvents, aliphatic hydrocarbons, hydrophilic agents, impact modifiers,such as rubber, epoxidized rubber, maleated rubber, Engage™, Loditer,and other such materials, hydrophobic agents, enzymes, PHA miscibleagents, pigments, stabilizers, and/or rubbers. In additionalembodiments, copolymers and/or other compatibilizers comprise randomcopolymer polypropylene or other polymers that include two or moremonomers, wherein one of the polymer of monomer groups may have a highermiscibility or compatibility than PHA than the other polymer or monomer.In several embodiments, the additional of one or more of such non-PHApolymer or material advantageously improves the post-production handlingof PHA. In one embodiment, PHA, such as PHB or PHBV, is mixed with oneor more of PP, PE, PVC, ABS, PS, TPU, PET, HIPS, BOPP, PP (randomcopolymer), PP (homopolymer), PP (copolymer), PP (clarified), PE (linearlow density), PE (low density), PE (high density), PS (high impact), PS(crystalline), PS (semi-crystalline), wherein the concentration of PHBis about 0.1 to about 99.9%, including about 1%, about 3%, about 5%,about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about30%, about 50%, about 70%, about 90%, about 99.9%, or percentagesbetween these percentages, wherein an impact modifier is added at about0.01% to about 99%, including about 0.01%, about 2%, about 0.1%, about5%, about 20%, about 90%, or percentages between these percentages, apigment or pigment altering substance, including a clarifying pigment,such as blue, red, yellow, green, white, or clear nucleation (to provideclarity), a filler, fiber, or composite-generating material (such aswood fiber, rubber, calcium carbonate, glass, glass fiber, long glassfiber), and/or one or more antioxidant, stabilizer, plasticizer, orcompatibilizer. In one embodiment, a compound is provided comprising, byweight, about 88% PP (random copolymer), about 9% PHB (molecular weightbetween approximately 1000 and approximately 5,000,000 Daltons, whereinmolecular weight may be adjusted to improve the compatibility of PHBwith PP, or improve the clarity of the compound, or cause PHB to act asa plasticizer for the PP phase), wherein one advantage of thispercentage is that it does not impact the recyclability and/orbiodegradability of the compound, about 2% Engage impact modifier, andabout 1% clarifying (nucleating) pigment. In one embodiment, a compoundis provided comprising, by weight, about 87% PP (random copolymer),about 10% PHB (molecular weight between approximately 1000 andapproximately 5,000,000 Daltons, wherein molecular weight may beadjusted to improve the compatibility of PHB with PP, or improve theclarity of the compound, or cause PHB to act as a plasticizer for the PPphase), wherein one advantage of this percentage is that it does notimpact the recyclability and/or biodegradability of the compound, about2% Engage impact modifier, and about 1% clarifying (nucleating) pigment.In one embodiment, the molecular weight of the PHA, including PHB or PHBV, is specifically tailored to between about 100 and about 3,000,000Daltons to provide optimal clarity, functionality, and/or compatibilitywith the non-PHA material, such as PP, PE, PVC, ABS, PET, fillers,pigments, impact modifiers, plasticizers, or other functionalmodification materials. In one embodiment, the molecular weight of PHAis controlled by inducing a heat treatment step to reduce molecularweight and thereby improve the compatibility of PHA with othermaterials, including PHA and non-PHA materials. In another embodiment,PHA is functionally enhanced by increasing the polydispersity of the PHAmaterial to above about 1 and less than about 5, thereby causing the PHAto exhibit reduced crystallinity and/or improved performance and/orcompatibilization with PHA or non-PHA materials by causing parts of thePHA to act as self-plasticizer, self-nucleator, self-compatibilizer(e.g., improving the strength of the bonds between the PHA molecules),self-impact modifier, and/or plasticizer, nucleator, compatibilizer, orimpact modifier.

In additional embodiments, the clarity of PHA is increased through afunction of directed or passive crystal orientation, whereby crystalsmay be oriented parallel to or perpendicular from, for example, a filmprocessing extrusion die. In one embodiment, a biaxial orientationmachine used for the production of biaxially oriented polypropylene maybe employed to produce high clarity PHA films or other materials. In oneembodiment, the crystals of PHA may also be oriented to increase clarityin an extruder and/or pelletization system via directed stretching,cooling, chemical treatment, electrical treatment, sonic treatment, orother treatment that causes PHA crystals to align in such a manner thatless or smaller crystals are formed, or the crystal formations enablegreater material flexibility. In an additional embodiment, the clarityand/or miscibility of PHA with other PHAs or non-PHA polymers isincreased by reducing the molecular weight of the PHA and/or increasingthe polydispersity of PHA. In one embodiment, the molecular weightand/or polydisperity of PHA is lowered (e.g., from about 500,000 Daltonsto less than about 100,000 Daltons) to increase the clarity and/ormiscibility of PHA. Without being limited by theory, PHA with low MW orhigh polydispersity can surprisingly act as a self-nucleating and/orself-plasticizing material, which yields surprisingly useful functionalimprovements, such as increased clarity, increased flexibility,increased miscibility, increased plasticity, and increased toughness.

If incorporating biomass, the biomass may be present at variedconcentrations, depending on the embodiment. For example in oneembodiment, the biomass is present in the compound at a concentration ofmore than about 0.001%, while in other embodiments, the biomass ispresent in the mixture at a concentration of more than about 0.01%. Instill other embodiments, depending on the make-up of the biomass and thecharacteristics of the PHA, greater or lesser concentrations may also beused. Advantageously, a variety of types of biomass may be used, forexample, one or more of methanotrophic, autotrophic, and heterotrophicbiomass are used in several embodiments.

Also provided for herein is a method for the synthesis of apolyhydroxyalkanoate (PHA) in a biomass material, comprising the stepsof: (a) providing a medium comprising a biomass capable of metabolizinga source of carbon, and (b) increasing the concentration of one or morenutrients in the medium to cause the biomass to synthesize PHA orincrease the synthesis rate of PHA relative to the synthesis rate ofnon-PHA material.

Advantageously, a variety of types of biomass may be used, for example,one or more of methanotrophic, autotrophic, and heterotrophic biomassare used in several embodiments.

In several embodiments, the step of increasing the concentration of anutrient in the medium causes a reduction in the rate of production ofnon-PHA biomass relative to the rate of production of the PHA in thebiomass. This relative increase in efficiency (e.g., the reduction ofmetabolic resources spent producing non-PHA biomass) advantageouslyincreases the PHA production capacity of the biomass material (e.g., byincreasing the overall output, increasing the per unit biomass output orconcentration).

In several embodiments, the nutrient is selected from the groupconsisting of: ethylenediaminetetraacetic acid (EDTA), citric acid,iron, copper, magnesium, manganese, zinc, chromium, nickel, boron,molybdenum, calcium, potassium, boron, methane, phosphorus, oxygen,nitrogen, carbon dioxide and combinations thereof.

There is also provided a method for the synthesis of apolyhydroxyalkanoate (PHA) in a biomass material, comprising the stepsof: (a) providing a medium comprising a biomass and one or morenutrients, and (b) increasing the concentration of one or more of thenutrients in the medium to cause the biomass to metabolically synthesizePHA by using the biomass as a source of carbon for the production of thePHA. In several embodiments, the nutrient is selected from the groupconsisting of magnesium, iron, copper, zinc, nickel, chromium,phosphorus, oxygen, calcium, methane, carbon dioxide, hydroxyl ions,hydrogen ions, sulfate, nitrogen, and combinations thereof. Varioustypes of biomass may be used, for example, one or more ofmethanotrophic, autotrophic, methanogenic and heterotrophic biomass areused in several embodiments

Complementary to the methods, processes, and systems for PHA production,there is also provided herein a process for extracting apolyhydroxyalkanoate from a PHA-containing biomass slurry comprisingPHA, non-PHA biomass, and a liquid, comprising: a) controlling thepressure and temperature of the liquid to cause the non-PHA biomass tobecome soluble in the liquid, and b) reducing the amount of the liquidin the slurry.

Depending on the embodiment, the liquid in the slurry may be one or moreof methanol, water, carbon dioxide, or another a solvent, super criticalfluid, polymer, additive, plasticizer, and/or other fluid (e.g.,methylene chloride, acetone, methanol, sodium hypochlorite,hypochlorite, chloroform, or dichloroethane). Various pressures areused, depending on the embodiment, including pressure above atmosphericpressure and pressure below atmospheric pressure. In still additionalembodiments, phases or sequential changes between sub-atmospheric andsupra-atmospheric pressures are used. In conjunction with the pressure,temperatures may be varied, in some embodiments, ranging between 0 and250 degrees Celsius. In other embodiments, a broader range oftemperatures is used, for example −40 degrees Celsius to about 500degrees Celsius, including about −20, about −4, about 4, about 20, about37, about 100, about 150 about 200, about 300, or about 400 degreesCelsius. In one embodiment, the temperature is between 0 and 200 degreesCelsius and the pressure is between −30 mmHg and 30,000 psi. In severalembodiments, temperatures are maintained between 0 and 400 degreesCelsius, 20 and 80 degrees Celsius, 10 and 100 degrees Celsius, 5 and200 degrees Celsius, 100 and 200 degrees Celsius, or 0 and 300 degreesCelsius. In several embodiments, different combinations of temperatureand pressure may be used, depending on the makeup of the slurry, theconcentration of PHA in the slurry and other factors. Also depending onthe characteristics of the slurry and other factors, the step ofreducing the amount of the liquid in the slurry comprises reducing theamount of the liquid in the biomass slurry before, during, and/or afterthe step (a).

Reduction in the amount of the liquid can be achieved in a variety ofways, used either alone or in combination (e.g., sequentially) such asfor example, centrifugation, filtration, distillation, spray drying,flash drying, lyophilization, air drying, and/or oven drying. Theseprocesses can reduce the amount of liquid in the slurry by about 0.1 toabout 100%. In several embodiments, the reduction is over 50%, over 90%,or over 95%. Which method is chosen depends, in some embodiments, on thestate of the slurry at that time. Some methods are more efficient atremoval of liquid that others, and as such, may be better suited toslurries with a higher liquid content.

In some, embodiments, it may be necessary to increase the amount ofliquid in the slurry (e.g., to effect a more efficient downstream step),which depending on the embodiment can be done before, during, and/orafter the step (a). Various amounts of liquid may be added, depending onthe embodiment, for example such that the solids concentration in theslurry of less than about 1 g/L, less than about 5 g/L, less than about100 g/L, less than about 250 g/L, less than about 500 g/L, less thanabout 750 g/L, less than about 1000 g/L. or about 1300 g/L. In someembodiments, supercritical (SC) fluids, such as SC-CO₂ or SC-water areused to purify PHA, such that proteins and/or non-PHA materials arerendered at least partially solubilized in SC-CO₂, SC-water, hightemperature or high pressure water, and/or mixtures thereof. In someembodiments, compatibilizing extraction agents may be used, such asnon-PHA polymers that maintain miscibility with PHA and high solubilityin SC-fluids, such that the PHA, miscible polymer, and SC-fluid producea low viscosity solution allowing separation of PHA from non-PHAmaterial.

There is also provided a method for improving the functionalcharacteristics of a polyhydroxyalkanoate (PHA) material, comprising thesteps of: (a) providing a PHA, a biomass, and a non-PHA polymer, (b)combining the PHA, the non-PHA polymer, and the biomass in a mixture toform a compound, (c) heating the compound to between about 100 degreesCelsius and about 250 degrees Celsius. Depending on the embodiment, thebiomass is present in the mixture at a concentration of between about0.1 and about 0.8%, between about 0.1 and about 20%, between about 0.1and about 40%, between about 0.1 and about 60%, between about 0.1 andabout 80%, and overlapping ranges thereof.

In several embodiments the biomass is methanotrophic biomass, while inother embodiments, the biomass is autotrophic biomass, heterotrophicbiomass, in combination, alone or with methanotrophic and/ormethanogenic biomass.

In several embodiments, the non-PHA polymer is one or more of thefollowing: polypropylene, polyethylene, polystyrene, polycarbonate,acrylonitrile butadiene styrene, polyethylene terephthalate, polyvinylchloride, fluoropolymers, liquid crystal polymers, acrylic,polyamide/imide, polyarylate, acetal, polyetherimide, polyetherketone,nylon, polyphenylene sulfide, polysulfone, cellulosics, polyester,polyurethane, polyphenylene oxide, polyphenylene ether, styreneacrylonitrile, styrene maleic anhydride, thermoplastic elastomer, ultrahigh molecular weight polyethylene, epoxy, melamine molding compound,phenolic, unsaturated polyester, polyurethane isocyanates, urea moldingcompound, vinyl ester, polyetheretherketone, polyoxymethylene plastic,polyphenylene sulfide, polyetherketone, polysulphone, polybutyleneterephthalate, polyacrylic acid, cross-linked polyethylene, polyimide,ethylene vinyl acetate, styrene maleic anhydride, styrene-acrylonitrile,poly(methyl methacrylate), polytetrafluoroethylene (including PTFE thatis modified to increase miscibility with a PHA, such as a PHB or PHBV,including PTFE with molecular weight between about 1 and about 5,000,about 1 and about 10,000, about 5 and about 50,000, or above about50,000), polybutylene, (including polybutylene that is modified toincrease miscibility with a PHA, such as a PHB or PHBV, includingpolybutylene with molecular weight between about 1 and about 5,000,about 1 and about 10,000, about 5 and about 50,000, or above about50,000), polylactic acid (including polylactic acid that is modified toincrease miscibility with a PHA, such as a PHB or PHBV, including PLAwith molecular weight between about 1 and about 5,000, about 1 and about10,000, about 5 and about 50,000, or above about 50,000), polyvinylchloride (including polyvinyl chloride that is modified to increasemiscibility with a PHA, such as a PHB or PHBV, including PVC withmolecular weight between about 1 and about 5,000, about 1 and about10,000, about 5 and about 50,000, or above about 50,000), polyvinylacetate (including polyvinyl acetate that is modified to increasemiscibility with a PHA, such as a PHB or PHBV, including PVAc withmolecular weight between about 1 and about 5,000, about 1 and about10,000, about 5 and about 50,000, or above about 50,000), polyvinylacetate co-polyvinylpyrrolidone (including Polyvinyl acetateco-Polyvinylpyrrolidone that is modified to increase miscibility with aPHA, such as a PHB or PHBV, including Polyvinyl acetateco-Polyvinylpyrrolidone with molecular weight between about 1 and about5,000, about 1 and about 10,000, about 5 and about 50,000, or aboveabout 50,000), polyvinylpyrrolidone (including Polyvinylpyrrolidone thatis modified to increase miscibility with a PHA, such as a PHB or PHBV,including Polyvinylpyrrolidone with molecular weight between about 1 andabout 5,000, about 1 and about 10,000, about 5 and about 50,000, orabove about 50,000), polyvinyl alcohol (including Polyvinyl alcohol thatis modified to increase miscibility with a PHA, such as a PHB or PHBV,including Polyvinyl alcohol with molecular weight between about 1 andabout 5,000, about 1 and about 10,000, about 5 and about 50,000, orabove about 50,000), cellulose, lignin, cellulose acetate butryate,polypropylene, polypropylene carbonate, propylene carbonate,polyethylene, ethyl alcohol, ethylene glycol, ethylene carbonate,glycerol, polyethylene glycol, pentaerythritol, polyadipate, dioctyladipate, triacetyl glycerol, triacetyl glycerol-co-polyadipate,tributyrin, triacetin, chitosan, polyglycidyl methacrylate, polyglycidylmethacrylate, oxypropylated glycerine, polyethylene oxide, lauric acid,citric acid, trilaurin, citrate esters, triethyl citrate, tributylcitrate, acetyl tri-n-hexyl citrate, saccharin, boron nitride, thymine,melamine, ammonium chloride, talc, lanthanum oxide, terbium oxide,cyclodextrin, organophosphorus compounds, sorbitol, sorbitol acetal,sorbitol-based nucleating agent, sorbital-like nucleating agent, sodiumbenzoate, clay, nanoclay, calcium carbonate (including calcium carbonatethat is included at various particle sizes or particle distributionsizes, including calcium carbonate that is included at very smallparticle size and optionally introduced at various points in anextrusion process, including (but not limited to) mostly at thebeginning, middle, or end, to minimize the processing time of thecalcium carbonate), sodium chloride, titanium dioxide, metal phosphate,glycerol monostearate, glycerol tristearate, 1,2-hydroxystearate,cellulose acetate propionate, polyepichlorohydrin, polyvinylphenol,polymethyl methacrylate, polyvinylidene fluoride, polymethyl acrylate,polyepichlorohydrin-co-ethylene oxide, polyvinyl idenechloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, and/or polyvinylidene chloride.

There is also provided, in several embodiments, a method for thesynthesis of polyhydroxyalkanoate (PHA) in a biomass material,comprising providing a medium comprising a biomass metabolizing a sourceof carbon, and increasing or maintaining above a minimum, theconcentration of an element in the medium to cause the biomass tosynthesize PHA and/or increase the synthesis rate of PHA relative to thesynthesis rate of non-PHA material. In several embodiments the PHA ispolyhydroxybutyrate (PHB), while in other embodiments other types of PHAare produced. In several embodiments, the biomass is one or moremicroorganisms. In one embodiment, the biomass comprises one or morerecycled microorganisms (e.g., those that have already been processedthrough a PHA synthesis process). In one embodiment, the microorganismshave been processed to remove at least a portion of the PHA theyproduced in the prior synthesis process.

In several embodiments, the increase in the concentration of an elementin the medium above a minimum concentration (or maintenance above thatminimum) causes a reduction in the concentration or production of sugar,lipids, nucleic acids, saccharides, polysaccharides, methanobactin,and/or pigments in the biomass relative to the concentration orproduction of PHA in the biomass. In several embodiments, the elementthat is increased or maintained is one or more of phosphorus, oxygen, ornitrogen. In additional embodiments, the element is one or more of EDTA,citric acid, iron, copper, magnesium, manganese, zinc, calcium,potassium, boron, methane, or carbon dioxide.

In still additional embodiments, the PHA synthesis rate is increasedrelative to the synthesis rate of PHA in the biomass in the absence ofthe increase or maintenance above a minimum the concentration of anelement in the medium.

There is also provided a method for the synthesis ofpolyhydroxyalkanoate (PHA) in a biomass material, comprising the stepsof: (a) providing a medium comprising a biomass and an element, and (b)maintaining above a minimum concentration or increasing theconcentration of the element in the medium to cause the biomass materialto metabolically synthesize PHA at the expense of alternative biomassenergy and/or carbon storage materials.

In several embodiments, the element that is increased (or maintained) isone or more of phosphorus, oxygen, magnesium, calcium, copper, iron,methane, carbon dioxide, hydroxyl ions, hydrogen ions, or nitrogen. Insome embodiments, the amount of increase (or maintenance) that isrequired of one element is altered when more than one element ismanipulated. For example, if phosphorous is increased in combinationwith another element, the overall amount of phosphorous needed isreduced as compared to the addition of phosphorous alone (e.g., thecombination of elements potentiates the effect).

In several embodiments, the biomass comprises one or moremicroorganisms, such as for example, methanotrophic microorganisms,heterotrophic microorganisms, autotrophic microorganisms, methanogenicmicroorganisms, or combinations thereof.

More specifically, certain embodiments of the invention provide highefficiency, high density, high PHA concentration processes for theproduction of PHA from carbon-containing gases, comprising the steps of:(a) providing a microorganism culture comprising PHA-containing biomass,(b) removing a portion of the PHA-containing biomass from the culture,(c) extracting a portion of PHA from the removed culture to produceisolated PHA and PHA-reduced biomass, (d) purifying the isolated PHA,and (e) returning the PHA-reduced biomass to the culture to cause theculture to convert the carbon within the PHA-reduced biomass into PHA.In several embodiments, carbon output from the system is wholly orsubstantially only in the form of PHA.

In several embodiments, a system for using a microorganism culture toconvert a carbon-containing gas into PHA at high efficiencies isprovided. Microorganisms are cultured using a combination of one or morecarbon-containing gases and PHA-reduced biomass, or derivatives thereof,as sources of carbon to produce PHA-containing biomass. A portion of thePHA-containing biomass is then removed from the culture, and PHA isextracted from the removed PHA-containing biomass to createsubstantially PHA-reduced biomass and substantially isolated PHA.

Typically, PHA is present in the PHA-containing biomass of gas-utilizingmicroorganisms at concentrations in the range of about 5%-60%, andapproximately 40-95% of the PHA-containing biomass is discarded from thesystem following PHA extraction. In some cases, PHA is present ingas-utilization microorganisms in the range of about 1-90%, including atabout 1%, about 3%, about 5%, about 7%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about60%, about 70%, about 80%, or about 90%, and approximately 10-99% of thePHA-containing biomass is discarded from the system following PHAextraction, including about 99%, about 97%, about 95%, about 93%, about90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%,about 55%, about 50%, about 40%, about 30%, about 20%, or about 10% ofthe PHA-containing biomass. Rather than discarding the remaining, e.g.,40-95% of the PHA-reduced biomass, in one embodiment of the invention,the PHA-reduced biomass is returned back to the microorganism culture tobe regenerated as PHA by a microorganism culture capable of utilizingPHA-reduced biomass, or a derivative thereof, as a source of carbon forPHA production, thereby creating a regenerative closed-looppolymerization system. By using PHA-reduced biomass as a source ofcarbon for PHA production in microorganisms growing as or in associationwith gas-utilizing microorganisms, PHA can be produced fromcarbon-containing gases at surprisingly and unexpectedly improvedcarbon, energy, and chemical efficiencies, since carbon fromcarbon-containing gases that would otherwise be discarded is regeneratedas PHA in a microorganism culture, and microorganisms that produce PHAfrom carbon-containing gases at low concentrations (e.g., 5-60% PHA byweight, or less than 70% PHA by weight) can, in some embodiments, beutilized to produce PHA at significantly increased carbon-to-PHAefficiencies. In some embodiments, the regeneration step is repeated toform an essentially closed-loop system. Thus, in some embodiments, thecarbon output from the system is at least about 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% (or more) PHA In other words, atleast about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95%, 99% (ormore) of the carbon entering the system is converted into PHA. In otherembodiments, about 1-5%, about 5-10%, about 10-20%, about 20-30%, about30%-40%, about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%,about 80%-90%, about 90%-95%, about 95%-99% (and overlapping rangesthereof) of the carbon entering the system is converted to PHA. Byregenerating PHA-reduced biomass as PHA in a microorganism culture, thepercentage of carbon from a carbon containing gas that is converted toPHA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or greater than systems that do not employ theregenerative or closed-loop system disclosed herein. In someembodiments, the regeneration (e.g., return and/or recycling of thePHA-reduced biomass) step is repeated at least 2, 3, 4, 5, 6, 7, 8, 9,or 10 (or more) times. In some embodiments, the regeneration step isrepeated until at least 90% to 95% of the carbon input into the systemis converted into PHA. In some embodiments, the regeneration step isrepeated as many times as desired to reach a particular percentageconversion of carbon to PHA.

Several embodiments of the invention provide for the production of PHAfrom carbon-containing gases at previously unattainable energy, carbon,and chemical efficiencies by way of providing a microorganism culturecapable of metabolizing the carbon within both a carbon-containing gasand PHA-reduced biomass, manipulating the conditions of the culture tocause the culture to produce PHA, removing a portion of PHA-containingbiomass from the culture, extracting the PHA within the removedPHA-containing biomass to create substantially isolated PHA andsubstantially PHA-reduced biomass, returning the PHA-reduced biomass tothe culture and contacting the PHA-reduced biomass with the culture tocause the culture to metabolize the carbon within the PHA-reducedbiomass into PHA, and purifying the isolated PHA. Thus, an advantage ofseveral embodiments of the invention is the production of PHA fromcarbon-containing gases at significantly improved energy, carbon, andchemical efficiencies.

The process according to several embodiments disclosed herein yields arange of surprising benefits over current gas-based PHA productiontechnologies. To begin, whereas the cell density of gas-basedfermentation processes is traditionally limited by the mass transfer ordiffusion rates of one or more factors, such as light, oxygen, carbondioxide, methane, or volatile organic compounds, several embodimentsdisclosed herein enable the generation of cell densities thatsignificantly exceed cell densities attainable in other currentpractices (e.g., by more than 1%, 10%, 20%, 30%, 50%, 80%, 100% ormore), and thereby enables cost-efficient system mixing, aeration, heatcontrol, and dewatering. For example, current methane-based PHAproduction systems are known to be capable (based on cell morphology andmass transfer characteristics) of generating approximately 60 g/L ofbiomass with an overall PHA concentration of 55%, or 33 g/L PHA. Incontrast, in several embodiments of the invention, cell densities ofapproximately 135 g/L with an overall PHA concentration of 70%, or 94.5g/L PHA are generated in a methane-based PHA production system. In someembodiments, cell densities of approximately 10 g/L, 20 g/L, 30 g/L, 60g/L, 75 g/L, 100 g/L, 125 g/L, 135 g/L, 150 g/L or greater are achieved.In some embodiments, overall PHA concentration in such cultures rangesfrom approximately 1% to 20%, 20% to 30%, 30% to 55%, 55% to 60%, 65% to70%, 70% to 80%, 80% to 90%, or 95% or greater, (and overlapping rangesthereof) result. In several embodiments, such PHA concentration rangesrepresent significant, unexpected, and surprising improvements overtraditional processes, e.g., processes that are limited to low celldensities and/or PHA concentrations.

As a non-limiting example of the impact of this improvement on energyefficiency, the energy required, on an energy input-to-PHA output basis,to aerate, mix, and dewater a 135 g/L solution with a PHA concentrationof 70% by weight is 186% less than the energy required to aerate, mix,and dewater a 60 g/L microorganism solution comprising 40% PHA byweight. In several embodiments, variations in the energy efficiencygains based on the systems and processes disclosed herein may occur,depending on the culture conditions, the strain or organisms used, andthe concentration and/or purity of an initial gas stream or other carbonsource. In several embodiments, even modest increases in efficiency havesubstantial benefits. For example, the ability to efficiently use aninput gas having a low carbon concentration that would not otherwise beuseful in PHA production may prevent the release of such a gas into theenvironment and/or reaction of the gas with other atmospheric compounds,thereby reducing the adverse impact of the low carbon concentration gason the environment (e.g., destruction of ozone, greenhouse gas emission,pollution, etc.).

Additionally, whereas current gas-based PHA production systems producesignificant carbon losses as a result of the low PHA inclusionconcentrations of gas-utilizing microorganisms (e.g., a significantportion of carbon and energy input is lost as biomass), severalembodiments of the invention enable the generation of overall carboninput yield efficiencies approaching maximum substrate values; e.g.,100% carbon input-to-PHA yield, minus respiration and/or downstreamprocessing losses. In some embodiments, at least 5%, at least 10%, atleast 30%, at least 50%, at least 70%, or at least 90%, carboninput-to-PHA yield is achieved. It is one important advantage of severalembodiments of the invention that maximum carbon yield efficiencies areunexpectedly and surprisingly generated in a PHA production systememploying gas-utilizing microorganisms, and particularly, in someembodiments, in PHA production systems employing gas-utilizingmicroorganisms that produce low biomass and/or PHA inclusion densities.

In some embodiments, the microorganism culture is a mixed culture,comprising heterotrophic microorganisms, methanotrophic microorganisms,autotrophic microorganisms, bacteria, yeast, fungi, algae, orcombinations thereof. In other embodiments, the microorganism culturemay be one or more cultures (e.g., a plurality of cultures). In someembodiments, the cultures are grown in one or more bioreactors. In someembodiments, the bioreactors utilize one or more culture conditions,including both aerobic and anaerobic conditions. In some embodiments,the microorganism culture converts PHA-reduced biomass to methane in ananaerobic process and subsequently to PHA in an aerobic process, suchthat PHA-reduced biomass is first anaerobically metabolized to methaneand then used as methane to produce biomass and PHA in a methanotrophicculture.

In several embodiments, at least part of the microorganism culture is amixed culture capable of metabolizing carbon-containing gases, includingmethane, carbon dioxide, greenhouse gases, and/or various other volatileorganic compounds, into biomass and/or PHA. In some embodiments, themicroorganism culture comprises a two phase system of anaerobic andanaerobic/aerobic metabolism, whereby carbon-containing gas is producedin a first substantially anaerobic phase and subsequently converted intoPHA in a second phase, wherein the microorganism culture in the firstphase is substantially anaerobic and the culture in the second phase iseither anaerobic or aerobic, wherein the two phases may be operated inone single vessel or in multiple vessels.

In some embodiments, at least one or more of the microorganisms arecontacted with artificial and/or natural light during one or more stepsof the methods disclosed herein.

In some embodiments, at least one of more of the microorganisms iscontacted with dissolved oxygen during one or more steps of the methodsdisclosed herein.

In some embodiments, at least one of more of the microorganisms iscultured at atmospheric, sub-atmospheric, or above-atmosphericpressures.

In some embodiments, at least one of more of the microorganisms canutilize only a carbon-containing gas as a source of carbon.

In several embodiments, at least one or more of the microorganisms canutilize carbon derived from a PHA-reduced biomass as a source of carbon.In other embodiments, at least one or more of the microorganisms is aheterotrophic microorganism capable of converting PHA-reduced biomassinto, carbon dioxide, oxygen, biomass, and/or PHA.

In several embodiments, at least one or more of the microorganisms arecultured using carbon derived from both a carbon-containing gas and aPHA-reduced biomass.

In some embodiments, the microorganism culture is a pure culture. Insome embodiments, the cultures are maintained in semi-sterile or sterileconditions.

In some embodiments, the microorganism culture is a mixed, non-sterileculture, including a naturally equilibrating consortium ofmicroorganisms.

In several embodiments, the microorganism culture is at least partiallycomprised of genetically engineered microorganisms.

In some embodiments, the microorganism culture is a mixed culturecomprising a combination of naturally occurring and geneticallyengineered microorganisms.

In several embodiments, the PHA is removed from the microorganismculture by solvent extraction, including solvent extraction attemperatures ranging from 0° C. to 200° C. and at pressures ranging from−30 psi to 30,000 psi.

In several embodiments, the PHA is removed from the microorganismculture through the utilization of ketones, alcohols, and/or chlorinatedsolvents.

In several embodiments, the PHA is removed from the microorganismculture by hypochlorite digestion and/or chlorine-based solventextraction.

In several embodiments, the PHA is removed from the microorganismculture by supercritical carbon dioxide extraction.

In several embodiments, the PHA is removed from the microorganismculture by protonic non-PHA cell material dissolution.

In several embodiments, the PHA is partially removed from themicroorganism culture to create a PHA-rich phase and a PHA-poor phase.

In several embodiments, the PHA is removed from the microorganismculture to render the PHA substantially free of non-PHA material,including substantially 5%, 10%, 20%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80-90%, 90-99% or more pure PHA by weight.

In several embodiments, the PHA is removed from the microorganismculture by manipulating the pH of the microorganism culture.

In certain embodiments, at least one or more of the microorganisms arecontacted with methane, carbon dioxide, oxygen, and/or a combinationthereof.

In several embodiments, multiple culture vessels are employed, such thatmicroorganism growth, PHA synthesis, PHA-reduced biomass metabolism, andPHA removal are carried out in separate vessels.

In other embodiments, microorganism growth, PHA-reduced biomassmetabolism, and PHA synthesis occurs in a single vessel.

In still other embodiments, microorganism growth and PHA synthesis occurin a single vessel and PHA extraction is carried out in one or moreseparate vessels.

In several embodiments of the process as disclosed herein, PHA synthesisis regulated by manipulating, increasing, decreasing, maintaining belowa maximum threshold, or maintaining above a minimum threshold theconcentration of a material in the process, wherein the material is oneor more of oxygen, methane, carbon dioxide, nitrogen, phosphorus,copper, iron, manganese, carbon, magnesium, potassium, cobalt, aluminum,sulfate, chlorine, boron, citric acid, and EDTA.

In several embodiments, the microorganism culture comprises one or morestrains of microorganisms collectively capable of converting the carbonwithin a carbon-containing gas into cellular biomass and the carbon fromcellular biomass or methane into PHA.

In several embodiments, the microorganisms are subjected to filtration,centrifugation, settling, and/or density separation.

In several embodiments, the isolated PHA and/or the PHA-reduced biomassis subjected to filtration, centrifugation, settling, and/or densityseparation.

In some embodiments, the processes disclosed herein further comprisewashing the recovered PHA with water or other liquid-based agents orsolvents to purify the PHA.

In several embodiments, the process further comprises oxidizing therecovered PHA to purify the PHA.

In several embodiments, the process further comprises drying therecovered PHA to remove volatiles such as water and/or one or moresolvents.

In several embodiments of the invention, methods for the production ofPHA are provided. In one embodiment, the method comprises: (a) providinga microorganism culture comprising PHA-containing biomass, (b) removinga portion of the PHA-containing biomass from the culture, (c) extractinga portion of the PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass, (d) returning the PHA-reducedbiomass to the culture to cause the culture to convert the carbon withinthe PHA-reduced biomass into PHA, and (e) purifying the isolated PHA.

In one embodiment, the microorganism culture utilizes the PHA-reducedbiomass, or derivatives thereof, such as carbon dioxide, methane, orvolatile organic acids, volatile fatty acids, volatile organiccompounds, non-methane organic compounds, and one or morecarbon-containing gas as a source of carbon. In one embodiment, the gasis selected from the group consisting of methane, carbon dioxide,volatile organic compounds, hydrocarbons, and combinations thereof. Inone embodiment, the gas is derived from one or more sources from thegroup consisting of: landfills, wastewater treatment plants, powerproduction facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, cement production facilities, and/oranaerobic organic waste digesters.

In some embodiments, the carbon in the PHA-reduced biomass is derivedfrom one or more gases from the group consisting of: methane, biogas,carbon dioxide, volatile organic compounds, natural gas, wastewatertreatment methane and VOCs, and hydrocarbons.

In some embodiments, natural and/or artificial light is utilized toinduce the metabolism of the carbon dioxide by the culture.

In some embodiments, the microorganism culture comprises one strain, ora consortium of strains, of microorganisms, including one or moremicroorganisms selected from the group consisting of: bacteria, fungi,yeast, and algae, and combinations thereof.

In some embodiments, the microorganism culture comprises one or moremicroorganisms from the group consisting of: methanotrophicmicroorganisms, carbon-dioxide utilizing microorganisms, anaerobicmicroorganisms, methanogenic microorganisms, acidogenic microorganisms,acetogenic microorganisms, heterotrophic microorganisms, autotrophicmicroorganisms, cyanobacteria, and biomass-utilizing microorganisms, andcombinations thereof.

In some embodiments, at least a portion of the microorganism culture isnaturally occurring. In some embodiments, at least a portion of themicroorganism culture is genetically engineered. In some embodiments,naturally occurring and genetically engineered microorganisms are bothused in the culture. As used herein, the term genetically engineeredshall be given its ordinary meaning and shall also refer tomicroorganisms which have been manipulated to contain foreign (e.g., notfrom that microorganism) genetic material and/or foreign proteins. Incertain embodiments, microorganisms are genetically manipulated toexpress one or more enzymes or enzymatic pathways useful in PHAproduction. In certain embodiments, microorganisms are geneticallymanipulated to express a marker, enzyme, or protein useful allowing theselective identification of the genetically engineered microorganisms.

In some embodiments, the microorganism culture is at least partiallymaintained under above-atmospheric pressure.

In some embodiments, the PHA-containing biomass includes one or moremicroorganism-derived materials selected from the group consisting of:intracellular, cellular, and/or extracellular material, including apolymer, amino acid, nucleic acid, carbohydrate, lipid, sugar,polyhydroxyalkanoate, chemical, and/or metabolic derivative,intermediary, and/or end-product. In some embodiments, thePHA-containing biomass includes one or more microorganism-derivedmaterials selected from the group consisting of: methane, volatileorganic compounds, carbon dioxide, and organic acids.

In one embodiment, the PHA-containing biomass contains less than about95% water, including less than about 90%, about 85%, about 80%, about75%, or about 70% water.

In some embodiments, the PHA-containing biomass is mixed with one ormore chemicals, including one or more chemicals from the groupconsisting of: methylene chloride, acetone, ethanol, methanol, ketones,alcohols, chloroform, and dichloroethane, or combinations thereof.

In one embodiment, the PHA-containing biomass is processed throughhomogenization, heat treatment, pH treatment, enzyme treatment, solventtreatment, spray drying, freeze drying, sonication, and microwavetreatment, or combinations thereof.

In one embodiment, the PHA-reduced biomass includes the PHA-containingbiomass wherein at least a portion of the PHA has been removed from thePHA-containing biomass. In another embodiment, the PHA-reduced biomassincludes methane, carbon dioxide, and organic compounds produced fromthe PHA-reduced biomass.

In some embodiments, the PHA-reduced biomass is subject to dewatering,chemical treatment, sonication, additional PHA extraction,homogenization, heat treatment, pH treatment, hypochlorite treatment,microwave treatment, microbiological treatment, including both aerobicand anaerobic digestion, solvent treatment, water washing, solventwashing, and/or drying, including simple or fractional distillation,spray drying, freeze drying, and/or oven drying, or combinationsthereof.

In several embodiments, the microorganism culture is maintained in asterile, semi-sterile, or non-sterile environment.

In one embodiment, the PHA includes one or more PHA selected from thegroup consisting of: polyhydroxybutyrate (PHB), polyhydroxyvalerate(PHV), polyhydroxybutyrate-covalerate (PHB/V), polyhydroxyhexanoate(PHHx), and short chain length (SCL), medium chain length (MCL), andlong chain length (LCL) PHAs.

In several embodiments, the metabolism, growth, reproduction, and/or PHAsynthesis of the culture is controlled, manipulated, and/or affected bya growth medium. In some embodiments, the bioavailable and/or totalconcentration of nutrients within the growth medium, such as copper,iron, oxygen, methane, carbon dioxide, nitrogen, magnesium, potassium,calcium, phosphorus, EDTA, calcium, sodium, boron, zinc, aluminum,nickel, sulfur, manganese, chlorine, chromium, molybdenum, and/orcombinations thereof are manipulated (e.g., increased, decreased, ormaintained) in order to control the metabolism, growth, reproduction,and/or PHA synthesis of the culture In some embodiments, a singlenutrient in the growth medium is manipulated, while in some embodiments,more than one nutrient in the growth medium is manipulated to achievethe desired effect on the culture. In several embodiments, manipulationof a nutrient (or other component of a culture medium) is performed, inseveral embodiments, in order to maintain the overall concentration ofthat nutrient in the medium over time within a certain desired range(e.g., if a nutrient is consumed by the microorganisms, a “replacement”amount of that nutrient is added to the medium).

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is induced and/or controlled by manipulating the composition of themedium. As discussed herein, the conversion of PHA-reduced biomass intothe PHA can be controlled in a time-dependent manner to maximize theefficiency of conversion. In some embodiments, conversion to PHAproduction is induced about 1-12 hours, about 5-15 hours, or about 8-24hours after PHA-reduced biomass is re-introduced into the culture. Insome embodiments, longer times, e.g., about 24 hours to several days orweeks, are employed.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is effected by manipulating the concentration one or more elementswithin a medium selected from the group consisting of: nitrogen,methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium, iron,copper, hydrogen, hydrogen ions, hydroxyl, hydroxyl ions, sulfate,manganese, calcium, chlorine, boron, zinc, aluminum, nickel, and/orsodium, and combinations thereof.

Methods used in several embodiments disclosed herein to control theconcentration of elements within the medium include, but are not limitedto, automatic, continuous, batch, semi-batch, manual, injection, solidfeed, liquid, or other methods of inputting one or more chemical intothe medium, wherein the total and/or bioavailable concentration ofelements is increased, decreased, maintained, adjusted, or otherwisecontrolled at one or more time and/or physical chemical adjustmentpoints.

According to several embodiments, additional methods to adjust the totalor bioavailable concentration of one or more elements within a mineralmedia include, but are not limited to, the directed precipitation,chelation, de-chelation, and de-precipitation of elements. In oneembodiment, the directed precipitation or chelation of one or moreelement is utilized to reduce the total or bioavailable concentration ofone or more element within a medium and thereby i) induce or increasePHA production in a biomass and/or ii) control the metabolism ofmicroorganisms within a medium, including for the purpose of controllingthe specification and/or functionality of PHA. In one embodiment,methanobactin is produced and/or utilized to chelate copper and/or ironin order to impact the metabolism of methanotrophic microorganisms.

In several embodiments, the concentration of one or more elements withina growth culture medium is increased, controlled, manipulated, orotherwise managed to induce or increase the rate of PHA production inbiomass, including a microorganism culture. In one embodiment, theconcentration of phosphorus within the medium is increased to induce orincrease the rate of PHA production in a microorganism culture. In oneembodiment, the concentration of an element, e.g., phosphorus, carbondioxide, iron, copper, oxygen, methane, hydroxyl ions, hydrogen ions,and/or magnesium, within the medium is manipulated or increased to causea metabolic shift in the microorganism culture, such that the productionof non-PHA materials by the culture using carbon and/or nitrogen sources(e.g., nitrate, ammonia, ammonium, dinitrogen, urea, or amino acids) isreduced, inhibited, or otherwise impacted to enhance PHA production. Inone embodiment, the concentration of phosphorus within the medium ismanipulated or increased to reduce the utilization of nutrients,including nitrogen, oxygen, and/or carbon, for the production of non-PHAmaterials by the culture. In one embodiment, the concentration ofphosphorus within the medium is manipulated or increased to reduce theutilization of nutrients for the production of non-PHA materials by theculture and induce or increase the rate of PHA production in theculture.

In several embodiments, an increase in the concentration of phosphoruscauses a metabolic shift that favors the production of PHA at theexpense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars,methanobactin, lipids, particularly but not necessarily undergrowth-limiting conditions, including nitrogen (e.g., nitrate, ammonia,ammonium, dinitrogen, urea, or amino acids), oxygen, magnesium,potassium, iron, copper, or other nutrient-limiting conditions.

In several embodiments, depending on the strain of microorganism, anincrease in the concentration of phosphorus above about 0.00 ppm, 0.01ppm, 0.02 ppm, 0.05 ppm, 0.10 ppm, 0.20 ppm, 0.50 ppm, 1.00 ppm, 1.25ppm, 1.50 ppm, 1.75 ppm, 2.00 ppm, 2.20 ppm, 2.40 ppm, 3 ppm, 4 ppm 5ppm, 6 ppm, 8 ppm, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90mM, 100 mM, 110 mM, 120 mM, 140 mM, 200 mM, 400 mM, 600 mM, 800 mM, or1000 mM, or overlapping ranges between these concentrations, causes areduction in the utilization of carbon and/or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,nucleic acids, lipids, pigments, polysaccharides, methanobactin, and/orcarbon dioxide, and, under growth-limiting conditions, an increase inthe utilization of carbon sources for the production of PHA material, asa result of metabolic changes in the culture and/or chemicalinteractions between chemicals within the media and/or culture inducedby augmented concentrations of phosphorus. The elevation of phosphorusconcentrations as a method to induce or increase the rate of PHAproduction in a biomass culture is contrary to the conventional wisdomin the field, which suggests that PHA production is induced or enhancedby reducing or eliminating the concentration of elements, such as, e.g.,phosphorus in the mineral medium. The addition or controlled elevationof an element, such as, e.g., phosphorus to a biomass system to induceor increase PHA production produces an unexpected and surprisingincrease in PHA production in a biomass system. An element such as,e.g., phosphorus may be added to the mineral media using a variety ofphosphorus sources, including phosphorus, phosphate, phosphoric acid,sodium phosphate, disodium phosphate, monosodium phosphate, and/orpotassium phosphate, dissolved carbon dioxide, Fe(II) iron, Fe(III)iron, copper sulfate, Fe-EDTA, dissolved oxygen, dissolved methane,and/or magnesium sulfate, among other potential sources. In oneembodiment, copper concentrations are manipulated or maintained above aminimum concentration, for at least a period of time, in order to reducethe concentration of methanobactin in the medium in order to increasethe purity of PHA produced by and/or extraction from a methanotrophicmicroorganism culture.

In several embodiments of the invention, the concentrations of dissolvedgases, such as methane, oxygen, carbon dioxide, and/or nitrogen, aremanipulated to increase the rate of PHA production relative to the rateof cellular production of non-PHA materials and, specifically, to causea reduction in the utilization of carbon or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,enzymes, nucleic acids, lipids, pigments, polysaccharides,methanobactin, and/or carbon dioxide, and, under some conditions,including growth-limiting conditions, further cause an increase in theutilization of carbon sources for the production of PHA material, as aresult of metabolic changes in the culture and/or chemical interactionsbetween chemicals within the media and/or culture induced by augmentedconcentrations of one or more of such dissolved gases. In oneembodiment, the concentration of methane or dissolved methane ismanipulated to above at least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1.0ppm, 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm,50 ppm, 100 ppm, 200 ppm, 300 ppm, 500 ppm, or 1000 ppm, or overlappingranges between these concentrations to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Insome embodiments, the concentration of oxygen or dissolved oxygen ismanipulated to above at least 0.0001 ppm, 0.01 ppm, 0.05 ppm, 0.1, 0.2ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1.0ppm, 1.1 ppm, 1.2 ppm, 1.3 ppm, 1.4 ppm 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5 ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm,500 ppm, 750 ppm, or 1000 ppm, or overlapping ranges between theseconcentrations to reduce the production of non-PHA materials relative tothe production of PHA materials in a culture. In one embodiment, theconcentration of carbon dioxide or dissolved carbon dioxide ismanipulated to at least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1.0 ppm,1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5 ppm,5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm, 50ppm, 100 ppm, 200 ppm, 500 ppm, 1000 ppm, 1500 ppm, 2000 ppm, 3000 ppm,5000 ppm, 10,000 ppm, 20,000 ppm, or overlapping ranges between theseconcentrations to reduce the production of non-PHA materials relative tothe production of PHA materials in a culture. In some embodiments, theconcentration of nitrogen or dissolved nitrogen is manipulated to aboveat least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1.0 ppm, 1.5 ppm, 1.75ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5 ppm, 5.0 ppm, 6.0ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm, or 50 ppm orranges between these concentrations to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inseveral embodiments, an increase in the concentration of methane,oxygen, carbon dioxide, and/or nitrogen causes a metabolic shift thatfavors the production of PHA at the expense of other non-PHA materials,including a reduction in the production of protein, nucleic acids,polysaccharides, sugars, and/or lipids, particularly, but notnecessarily, under growth-limiting, that is, PHA synthesis, conditions.

In some embodiments, the PHA is at least partially removed from thePHA-containing biomass or otherwise purified using one or moreextraction agents or mechanisms selected from the group consisting of:methylene chloride, acetone, ethanol, methanol, dichloroethane,supercritical carbon dioxide, sonication, homogenization, water, heat,distillation, spray drying, freeze drying, centrifugation, filtration,enzymes, polymers, surfactants, co-solvents, hydrolyzers, acids, bases,hypochlorite, peroxides, bleaches, ozone, EDTA, miscible agents, and/orcombinations thereof.

In one embodiment, the extraction process is substantially carried outat intracellular temperatures less than 100° C. In other embodiments,temperatures for extraction range from about 10° C. to 30° C., fromabout 30° C. to 50° C., from about 50° C. to 70° C., from about 70° C.to 90° C., from about 90° C. to about 120° C., from about 100° C. toabout 140° C., from about 20° C. to 150° C., or from about 120° C. toabout 200° C., or higher. In another embodiment, cells are reused forpolymerization following the extraction process as viable cells.

In some embodiments, PHA-containing biomass is treated with one or morechemical treatment steps to control, modify, or increase theconcentration or functional characteristics (e.g., molecular weight,monomer composition, melt flow profile, purity, non-PHA residualsconcentration, protein concentration, DNA concentration, antibodyconcentration, antioxidant concentration) of PHA in a PHA-containingmaterial or biomass.

As used herein, the terms “functional properties” and “functionalcharacteristics” shall be given their ordinary meanings and shall alsorefer to the specification, features, qualities, traits, or attributesof PHA. The functional characteristics of the generated PHA include, butare not limited to molecular weight, polydispersity and/orpolydispersity index, melt flow and/or melt index, monomer composition,co-polymer structure, melt index, non-PHA material concentration,purity, impact strength, density, specific viscosity, viscosityresistance, acid resistance, mechanical shear strength, flexularmodulus, elongation at break, freeze-thaw stability, processingconditions tolerance, shelf-life/stability, hygroscopicity, and color.As used herein, the term “polydispersity index” (or PDI), shall be givenits ordinary meaning and shall be considered a measure of thedistribution of molecular mass of a given polymer sample (calculated asthe weight average molecular weight divided by the number averagemolecular weight). Advantageously, several embodiments of the processesdisclosed herein may be carried out in sterile, semi-sterile, ornon-sterile conditions. In several embodiments, consistency in more thanone of these functional properties is achieved. For example, in someembodiments, consistent molecular weight, polydispersity, andcombinations thereof are achieved. In one embodiment, temperature isused to control, modify, reduce, or optimize the molecular weight,polydispersity, melt flow, and other characteristics of PHA. In oneembodiment, temperature and/or time is used to control the molecularweight of PHA between the range of about 5,000,000 and about 10,000Daltons. In several embodiments the molecular weight of PHA rangesbetween about 5,000,000 and about 2,500,000 Daltons, between about2,500,000 and about 1,000,000 Daltons, between about 1,000,000 and about750,000 Daltons, between about 750,000 and about 500,000 Daltons,between about 500,000 and about 250,000 Daltons, between about 250,000and about 100,000 Daltons, between about 100,000 and about 50,000Daltons, between about 50,000 and about 10,000 Daltons, and overlappingranges thereof. In one embodiment, a slurry comprising PHA-containingbiomass and a culture media is subject to one or more water removalsteps or water addition steps to increase the concentration of PHA in aPHA-containing biomass. In one embodiment, the water removal step is adewatering step or combination of dewatering steps, such ascentrifugation, filtration, spray drying, flash drying, and/or chemicaldewatering (e.g., with acetone, ethanol, or methanol, or combinationsthereof), wherein at least a portion of the water concentration relativeto the concentration of PHA-containing biomass in the slurry is reduced.In one embodiment, a temperature and/or pressure control step is carriedout under atmospheric (0 psi), sub-atmospheric (−100-0 psi), orabove-atmospheric pressure (e.g., 0-30,000 psi) and at temperatureconditions wherein the PHA-containing biomass, or the liquid in and/oraround the PHA-containing biomass, is maintained, for at least a periodof time, at a temperature ranging from −30 to 10 degrees Celsius, 10degrees Celsius to 100 degrees Celsius, 10 degrees Celsius to 150degrees Celsius, 20 degrees Celsius to 250 degrees Celsius, and/or 100to 200 degrees Celsius, including overlapping ranges thereof. In oneembodiment, the PHA-containing biomass is subject to a dewatering stepbefore or after the temperature and/or pressure control step, whereinthe dewatering step is centrifugation, filtration, and/or spray drying,to produce a fully or partially de-watered PHA-containing biomass orPHA-containing biomass slurry, wherein the water concentration of thedried slurry is less than 99%, 95%, 80%, 60%, 40%, 30%, 20%, 10%, 5%,3%, 2%, or 1% water. In one embodiment, the PHA-containing biomass issubject to a temperature control step, wherein the water or liquidchemicals within and/or around the biomass is controlled and maintainedat a temperature of at least −30, −10, −5, −4, −3, −2, −1, 0, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 degreesCelsius or ranges between those temperatures. In one embodiment, thePHA-biomass is not dried prior to such temperature control step. In oneembodiment, the PHA-containing biomass is dried or de-watered prior tosuch temperature control step. In one embodiment, the PHA-containingbiomass is filtered or centrifuged following the temperature controlstep. In one embodiment, the PHA-containing cell slurry is notdewatered, for example, by centrifugation or other drying mechanism,prior to the temperature control step. In one embodiment, a mechanism toimpart shear onto or into the PHA-containing biomass is coupled with atemperature control step; such shear may be imparted in the form of oneor more shear induction mechanisms including, but not limited to e.g., acentrifugal pump, agitator, blender, high shear mixer, vortex mixer,etc. In one embodiment, the PHA-containing biomass is dewatered in onestep and the treated PHA-containing biomass is further dewatered in oneor more additional steps. In one embodiment, the PHA-containing biomassis dewatered, water and/or other chemicals are added and temperatureand/or pressure is controlled, and the treated PHA-containing biomass isfurther dewatered and/or purified. In one embodiment, the water and/orchemicals within, around, and/or added to the PHA-containing biomass istemperature and/or pressure controlled, and the treated PHA-containingbiomass is further purified in one or more purification steps. In oneembodiment, the temperature control step process time is approximately 1second, 5 seconds, 10 seconds, 25 seconds, 60 seconds, 2 minutes, 5minutes, 20 minutes, 45 minutes, 1 hour, 2 hours, 5 hours, 6 hours, 7hours, 12 hours, 15 hours, 24 hours, 36 hours, or 48 hours, or rangesbetween those times. In one embodiment, inorganic materials may be usedto effect PHA modification, purification, or extraction, includingcarbon dioxide and dinitrogen. In one embodiment, the PHA-containingslurry or biomass is treated with carbon dioxide under elevatedtemperatures and pressures, including supercritical ranges, to inducePHA extraction or functional modification of PHA. In one embodiment,solvents, including methylene chloride, enzymes, super critical fluids,acetone, chloroform, dichloroethane, ethanol, plasticizers, acids,bases, polymers, and/or methanol are used, alone or in combination, inconjunction with any of the above steps to improve efficiency, includingincreasing purity, recovery, extractability, solubility, reaction speed,odor, color, recyclability, biodegradability, toxicity, endotoxinremoval rate, and the like. In one embodiment, solvents or extractionmaterials may be used or recycled for biomass production, biogasproduction, and/or PHA synthesis.

In one embodiment, chemicals are added to a PHA-containing biomass tocause the crystallization of PHA. In one embodiment, methylene chloride,carbon dioxide, acetone, water, dichloroethane, or methanol may be addedto a PHA-containing biomass in order to induce the crystallization ofPHA in the PHA-containing biomass. In some embodiments, this step may beuseful for the downstream processing of PHA, wherein crystallized PHA isless prone than amorphous PHA to degradation, including molecular weightloss, when contacted with extraction chemicals, including solvents,enzymes, acids, bases, and bleach. In one embodiment, silicon, silica,derivatives thereof, and/or chemicals containing silicon may be added tothe PHA-containing biomass in order to impact the metabolic status ofthe culture, and thereby control the functional characteristics of thePHA produced by the culture, including one or more of the following:molecular weight, monomer composition, co-polymer structure, melt index,and polydispersity.

In one embodiment, the removal of the PHA from the culture causes theculture to be temporarily deactivated, such that the culture, orelements thereof, may be further used for the synthesis of PHA. Incertain embodiments, deactivation is beneficial because it allows forthe delay of PHA production, transfer of material to another productionarea, and the like. In some embodiments, deactivation allows a tailoredPHA production time frame. In some embodiments, the reuse of cells forpolymerization is beneficial because it avoids or reduces the need toproduce new biomass prior to polymerization, thereby reducing thecarbon, chemical, and energy requirement of PHA production.

In one embodiment, a PHA produced according to the several embodimentsdescribed herein is provided.

In several embodiments, processes for the production of PHA from acarbon-containing gas are provided. In one embodiment, the processcomprises the steps of: a) providing a growth medium comprising amicroorganism culture capable of utilizing the carbon within one or morecarbon-containing gas and PHA-reduced biomass, b) manipulating themedium to cause the culture to produce PHA, c) removing at least aportion of the PHA within the culture to create substantially isolatedPHA and substantially PHA-reduced biomass, d) purifying the isolatedPHA, and e) returning the PHA-reduced biomass to the culture to causethe culture to metabolize the carbon within the PHA-reduced biomass intoPHA.

In one embodiment, the carbon-containing gas is selected from the groupconsisting of: methane, carbon dioxide, toluene, xylene, butane, ethane,methylene chloride, acetone, ethanol, propane, methanol, vinyl chloride,volatile organic compounds, hydrocarbons, and combinations thereof. Asdiscussed herein, in some embodiments, non-gaseous carbon-containingmaterial can be used, at least in part, for the production of PHA.

In one embodiment, the invention comprises manipulating theconcentration of elements, e.g., copper, iron, phosphorus, oxygen,methane, carbon dioxide, in the culture medium to control theconcentration of sMMO and/or pMMO produced by a methanotrophic culturein order to control the relative ratio of sMMO to pMMO in the culture,including over time and over multiple growth and polymerization cycles,and thereby control one or more of the growth conditions, metabolicstatus, metabolic disposition, and/or specification of PHA (e.g.,molecular weight, polydispersity, melt flow, monomer composition, etc.)produced by the culture, including over time. In some embodiments, sMMOis expressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingsMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout 100%, and overlapping ranges thereof. Simultaneously, orindependently, in some embodiments, pMMO is expressed in a range betweenabout 0% and 100% of a methanotrophic culture by dry cell weight, as apercentage of microorganisms expressing pMMO, or as a percentage oftotal MMO expressed by one or more methanotrophic cells, includingbetween 0% and 1%, between about 1% and about 2%, between about 2% andabout 3%, between about 3% and about 5%, between about 5% and about 10%,between about 10% and about 20%, between about 20% and about 30%,between about 30% and about 50%, between about 50% and about 70%,between about 70% and about 80%, between about 80% and about 90%,between about 90% and about 95%, between about 95% and about 100%, andoverlapping ranges thereof. In some embodiments, the ratio of sMMO topMMO produced in a methanotrophic culture is controlled to control thespecification of PHA produced by a culture. In some embodiments, therelative weight ratio of sMMO to pMMO in a methanotrophic culture is atleast or approximately 0 to 1, approximately 0.0000001 to 1,approximately 0.0001 to 1, approximately 0.001 to 1, approximately 0.01to 1, approximately 0.1 to 1, approximately 1 to 1, approximately 2 to1, approximately 3 to 1, approximately 5 to 1, approximately 10 to 1,approximately 15 to 1, approximately 20 to 1, approximately 25 to 1,approximately 30 to 1, approximately 35 to 1, approximately 50 to 1,approximately 65 to 1, approximately 70 to 1, approximately 80 to 1,approximately 90 to 1, approximately 95 to 1, approximately 98 to 1,approximately 99 to 1, approximately 100 to 1, approximately 1000 to 1,approximately 10,000 to 1, approximately 100,000 to 1, or approximately1,000,000 to 1, respectively. In some embodiments, the relative weightratio of pMMO to sMMO in a methanotrophic culture is approximately 0 to1, approximately 0.0000001 to 1, approximately 0.0001 to 1,approximately 0.001 to 1, approximately 0.01 to 1, approximately 0.1 to1, approximately 1 to 1, approximately 2 to 1, approximately 3 to 1,approximately 5 to 1, approximately 10 to 1, approximately 15 to 1,approximately 20 to 1, approximately 25 to 1, approximately 30 to 1,approximately 35 to 1, approximately 50 to 1, approximately 65 to 1,approximately 70 to 1, approximately 80 to 1, approximately 90 to 1,approximately 95 to 1, approximately 98 to 1, approximately 99 to 1,approximately 100 to 1, approximately 1000 to 1, approximately 10,000 to1, approximately 100,000 to 1, or approximately 1,000,000 to 1.

In some embodiments, by controlling the relative concentrations of sMMOand pMMO produced by a culture of methanotrophic microorganisms, it ispossible to control the metabolic status of the culture and therebycontrol the type (and characteristics) of PHA and other cellularmaterial produced by the culture, particularly in the presence of one ormore of the following: volatile organic compounds, fatty acids, volatilefatty acids, methanol, formate, acetone, acetate, acetic acid, formicacid, dissolved carbon dioxide, dissolved methane, dissolved oxygen,carbon-containing materials, ammonia, ammonium, and other elements orcompounds that impact the metabolism of a culture of methanotrophicmicroorganisms in a certain manner according to the relativeconcentration of sMMO or pMMO in such a culture. In some embodiments,sMMO and/or pMMO is expressed in a range between about 0% and 100% of amethanotrophic culture by dry cell weight, as a percentage ofmicroorganisms expressing sMMO or pMMO, or as a percentage of total MMOexpressed by one or more methanotrophic cells, including between 0% and1%, between about 1% and about 2%, between about 2% and about 3%,between about 3% and about 5%, between about 5% and about 10%, betweenabout 10% and about 20%, between about 20% and about 30%, between about30% and about 50%, between about 50% and about 70%, between about 70%and about 80%, between about 80% and about 90%, between about 90% andabout 95%, between about 95% and about or 100%, and overlapping rangesthereof prior to, during, throughout, or after a PHA production phase.

In one embodiment, sMMO is not expressed, or is expressed in low amounts(e.g., less than about 35%, about 25%, about 15%, about 5%, about 3% orabout 1%), in a methanotrophic culture prior to, during, throughout, orafter a PHA production phase. In some embodiments, the directed orcontrolled absence or reduction of sMMO in a methanotrophic cultureproducing PHA, particularly in the presence of non-methane organiccompounds that can be metabolized by methanotrophic microorganisms,engenders PHA production stability, consistency, and control byselectively shielding against the metabolism of one or some or manynon-methane organic compounds that might otherwise be metabolized in thepresence of sMMO, which enables the metabolism of a larger group ofnon-methane compounds than pMMO. Similarly, in one embodiment, pMMO isnot expressed, or is expressed in low amounts (e.g., less than about35%, about 25%, about 15%, about 5%, about 3% or about 1%), in amethanotrophic culture prior to, during, throughout, or after a PHAproduction phase. In some embodiments, the directed or controlledabsence or reduction of pMMO in a methanotrophic culture producing PHA,particularly in the presence of non-methane organic compounds that canbe metabolized by methanotrophic microorganisms, engenders PHAproduction stability, consistency, and control by selectively inducingor promoting the metabolism of one or some or many non-methane organiccompounds that might otherwise be metabolized using pMMO. Further, insome methanotrophic cultures, sMMO promotes PHA synthesis at highintracellular concentrations by reducing cellular production of non-PHAmaterials, particularly as compared to PHA synthesis using pMMO. Bycontrolling the concentration of sMMO relative to pMMO in amethanotrophic microorganism culture in the presence of methane and/ornon-methane organic compounds, including VOCs, volatile fatty acids,acetone, formate, ethane, propane, it is possible to control thespecification or type of PHA produced by the culture, including themolecular weight, polydispersity, and other similar functionalcharacteristics as disclosed herein. In some embodiments, it ispreferable to maintain the concentration of copper in the culture mediain order to promote sMMO production. In some embodiments, the productionof sMMO in many, most, or substantially all of the methanotrophic cellsenables the culture to produce more PHA when subject to a nutrientlimiting step than would otherwise be produced if the relative ratio ofpMMO in the culture was higher prior to the nutrient limiting step. Insome embodiments, it is preferable to maintain the concentration ofcopper in the culture media in order to promote pMMO production. In someembodiments, the production of pMMO in many, most, or substantially allof the methanotrophic cells enables the culture to produce more PHA whensubject to a nutrient limiting step than would otherwise be produced ifthe relative ratio of sMMO in the culture was higher prior to thenutrient limiting step. In one embodiment, one or more methanotrophiccells or cultures are subject to repeated growth and PHA synthesiscycles or steps, wherein the relative concentration of sMMO to pMMO inthe cells or cultures is controlled or caused to remain approximatelysimilar or substantially the same (e.g., within about 5% to about 10%,with about 10% to about 20%, within about 20% to about 30%, within about30% to about 40%, within about 40% to about 50%, within about 50 toabout 75%) or the same in each new cycle or step in order to control orkeep substantially the same (e.g., within about 5% to about 10%, withabout 10% to about 20%, within about 20% to about 30%, within about 30%to about 40%, within about 40% to about 50%, within about 50% to about75% across production runs) the specification, characteristics, and/orfunctionality (e.g., molecular weight, monomer composition, melt flowindex, polydispersity, non-PHA material concentration, and/or purity) ofthe PHA produced by or extractable from the culture or cultures in eachnew or repetitive cycle with the same or new cells.

In one embodiment, the invention comprises a PHA comprising carbonderived from PHA-reduced biomass, wherein the PHA-reduced biomasscomprises carbon derived from one or more carbon-containing gassesand/or one or more additional sources of carbon (either gaseous ornon-gaseous). In one embodiment, PHA is co-mingled and/or melted withPHA-reduced biomass to improve the functional characteristics of thePHA. In one embodiment, PHA is co-mixed and/or melted with PHA-removedbiomass and/or microorganism biomass to improve the functionalcharacteristics of PHA. In one embodiment, the percentage of non-PHAmicroorganism biomass included in a PHA, PHA compound, or PHA mixture isabout 0.00001% to about 0.001%, about 0.001% to about 0.01%, about 0.01%to about 0.1%, to about 0.1% to about 0.5%, about 0.5% to about 1%,about 1%, to about 2%, about 2% to about 3%, about 3% to about 5%, about5% to about 7%, about 7% to about 10%, about 10% to about 15%, about 15%to about 20%, about 20% to about 30%, about 30% to about 40%, about 40%to about 50%, about 50% to about 60%, about 60% to about 70%, about 70%to about 80%, about 80% to about 90%, about 90% to about 98%, about 98%to about 99.99%, and overlapping ranges thereof. In some embodiments,the inclusion of microorganism biomass to a PHA improves the functionalcharacteristics of a PHA by acting as one or more of the following:nucleating agent, plasticizer, compatibilizer, melt flow modifier, moldrelease agent, filler, strength modifier, elasticity modifier, ordensity modifier. In some embodiments, the microscopic size ofmicroorganism biomass, including nucleic acids and proteins, isparticularly and surprisingly effective as a functionalization agent forPHA. In some embodiments, microorganism biomass acts as a surprisinglyeffective compatibilizer for PHA and non-PHA polymers, such aspolypropylene and polyethylene. In one embodiment, the biomass issubject to a processing and/or modification step in order to make thebiomass, or at least a portion of the biomass, miscible with the PHAand/or non-PHA polymer, which yields unexpected and surprisingfunctional improvement of the biomass as a blend component. Suchprocessing/modification step may include: heat, shear, pressure, solventextraction, washing, filtration, centrifugation, sonication, enzymatictreatment, super critical material treatment, cellular dissolution,flocculation, acid and/or base treatment, drying, lysing, and/orchemical treatment, wherein said chemicals may include solvents, celldissolution agents, cell metabolizing agents, polymers, plasticizers,compatibilization agents, nucleating agents, including processing ormodification steps that enable PHA contained in said biomass to becomemiscible with said biomass and/or other materials, including a secondpolymer or carrier agent. In one embodiment, there is provided a methodfor modifying the functional characteristics of a polyhydroxyalkanoate(PHA) material, comprising the steps of: (a) providing a PHA and abiomass, (b) subjecting the PHA and/or the biomass to a processing andmodification step wherein the PHA and the biomass are subject totemperatures between at least about 20 degrees Celsius and about 250degrees Celsius and to pressures of at least between about 1 atmosphereand about 350 atmospheres, thereby causing the modified biomass toeffect a functional modification of the PHA material, wherein thefunctional modification comprises an increase in plasticization,nucleation, compatibilization, melt flow modification, strengthening,and/or elasticization. In one embodiment, the ratio of biomass to PHA insaid PHA material may be between about 1:1000 and about 1000:1,including between 1:1000 and 1:500, 1:500 and 1:100, 1:100 and 1:10,1:10 and 1:9, 1:9 and 1:8, 1:8 and 1:5, 1:5 and 1:3, 1:3 and 1:1, 1:1and 2:1, 2:1 and 5:1, 5:1 and 10:1, 10:1 and 100:1, 100:1 and 500:1, and500:1 and 1000:1, or ratios overlapping with the above ranges. In oneembodiment, the temperature, in degree Celsius, of the modification stepmay range from 20 to 40, 40 to 60, 60 to 80, 80 to 100, 100 to 120, 120to 140, 40 to 200, 140 to 160, 160 to 180, 180 to 200, 200 to 220, 220to 250, 250 to 300, 90 to 200, 140 to 220, and 50 to 400. In oneembodiment, the pressure, pounds per square inch, of the modificationstep may range from −30 to 50,000, −30 to 0, 0 to 50, 0 to 3,000, 0 to200, 0 to 10,000, 0 to 5,000, 0 to 20,000, 20,000 to 50,000, 10,000 to40,000, 25,000 to 30,000, 0 to 500, 0 to 1000, 0 to 2,000, and 40,000 to50,000. In one embodiment, the modification step may be used toeliminate the need for additional plasticization, nucleation,compatibilization, melt flow modification, strengthening, reduction ofPHA crystallinity or rate of crystallization, increase in opticalclarity, and/or elasticization. In one embodiment, microorganism biomassand/or modified biomass, as described above, is mixed with a PHA tomodify one or more of the nucleation, plasticization, compatibilization,melt flow, density, strength, elongation, elasticity, mold strength,mold release, and/or bulk density characteristics of a PHA, which may bemelted, extruded, film blown, die cast, pressed, injection molded, orotherwise processed. In one embodiment, PHA is partially or not removedfrom PHA-containing microorganism biomass prior to melt processing,e.g., extrusion, injection molding, etc. In one embodiment, non-PHAbiomass parts or materials are caused to remain with PHA derived from aPHA-containing biomass in order to modify or control the functionalcharacteristics of a PHA material. In one embodiment, PHA is present inPHA-containing biomass at a concentration ranging from 1-99.99999%,1-99.99%, 1-99%, 5-99%, 10-99%, 20-99%, 30-99%, 50-99%, 70-99%, 80-99%,90-99%, 95-99%, 98-99%, and overlapping ranges thereof. In oneembodiment, PHA, microorganism biomass, and/or one or more non-PHAmaterial, polymer, or thermoplastic are mixed, melted, or processedtogether. In one embodiment, the non-PHA polymer or material consists ofone or more of the following solvents, cell dissolution agents, cellmetabolizing agents, polymers, plasticizers, compatibilization agents,miscible agents, and nucleating agents: polypropylene, polyethylene,polystyrene, polycarbonate, acrylonitrile butadiene styrene,polyethylene terephthalate, polyvinyl chloride, fluoropolymers, liquidcrystal polymers, acrylic, polyamide/imide, polyarylate, acetal,polyetherimide, polyetherketone, nylon, polyphenylene sulfide,polysulfone, cellulosics, polyester, polyurethane, polyphenylene oxide,polyphenylene ether, styrene acrylonitrile, styrene maleic anhydride,thermoplastic elastomer, ultra high molecular weight polyethylene,epoxy, melamine molding compound, phenolic, unsaturated polyester,polyurethane isocyanates, urea molding compound, vinyl ester,polyetheretherketone, polyoxymethylene plastic, polyphenylene sulfide,polyetherketone, polysulphone, polybutylene terephthalate, polyacrylicacid, cross-linked polyethylene, polyimide, ethylene vinyl acetate,polyvinyl chloride, polyvinyl acetate, polyvinyl acetateco-polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinyl alcohol,cellulose, lignin, cellulose acetate butryate, polypropylene,polypropylene carbonate, propylene carbonate, polyethylene, ethylalcohol, ethylene glycol, ethylene carbonate, glycerol, polyethyleneglycol, pentaerythritol, polyadipate, dioctyl adipate, triacetylglycerol, triacetyl glycerol-co-polyadipate, tributyrin, triacetin,chitosan, polyglycidyl methacrylate, polyglycidyl methacrylate,oxypropylated glycerine, polyethylene oxide, lauric acid, trilaurin,citrate esters, triethyl citrate, tributyl citrate, acetyl tri-n-hexylcitrate, saccharin, boron nitride, thymine, melamine, ammonium chloride,talc, lanthanum oxide, terbium oxide, cyclodextrin, organophosphoruscompounds, sorbitol, sorbitol acetal, sodium benzoate, clay, calciumcarbonate, sodium chloride, titanium dioxide, metal phosphate, glycerolmonostearate, glycerol tristearate, 1,2-hydroxystearate, celluloseacetate propionate, polyepichlorohydrin, polyvinylphenol, polymethylmethacrylate, polyvinylidene fluoride, polymethyl acrylate,polyepichlorohydrin-co-ethylene oxide, polyvinyl idenechloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHB V, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, styrene maleic anhydride,styrene-acrylonitrile, poly(methyl methacrylate),polytetrafluoroethylene, polybutylene. polylactic acid, polyvinylidenechloride, and/or other similar materials or combinations of thesematerials, including mold release agents, plasticizers, solvents,solvent-grafted polymer, salts, nucleating agents, cross-linking agents,filaments, water, antioxidants, compatibilizers, co-polymers, peroxides,alcohols, ketones, polyolefins, chlorinated solvents, non-chlorinatedsolvents, aliphatic hydrocarbons, hydrophilic agents, hydrophobicagents, enzymes, PHA miscible agents, pigments, stabilizers, and/orrubbers. In several embodiments, the additional of one or more of suchnon-PHA polymer or material advantageously improves the post-productionhandling of PHA. In one embodiment, PHA, methanotrophic, autotrophic,and/or heterotrophic microorganism biomass, and a non-PHA polymer aremixed and melted together, including, in one embodiment, under elevatedpressures ranging from about 1 to about 5000 atmospheres. In oneembodiment, the concentration of non-PHA microorganism biomass in such amixture ranges from 0.0001% to 90%, 0.1% to 30%, 0.1% to 10%, or 0.5% to8%, and overlapping ranges thereof. In one embodiment, the concentrationof methanobactin in PHA is controlled to modify the functionalcharacteristics of the PHA, including color, odor, brittleness,flexibility, antioxidant activity, antiviral activity, and/orantibacterial activity. In one embodiment, reducing the concentration ofmethanobactin in the PHA reduces the brown or yellow shade of the PHAand increases the flexibility of the PHA. In another embodiment,increasing the concentration of methanobactin in the PHA increases theantimicrobial (e.g., antibacterial, antiviral) and/or antioxidantactivity or potential activity or the PHA. In one embodiment, a methodis provided for improving the functional characteristics of a PHApolymer through the melting and cooling of the PHA polymer in thepresence of a dual-functionalizing biomass agent and a second polymer,comprising the steps of: (a) providing a first polymer, a biomass, and asecond polymer, wherein the first polymer is a PHA, (b) subjecting thebiomass to a processing step comprising heat, pressure, solvent washing,filtration, centrifugation, super critical solvent extraction, and/orshear, wherein the processing step renders at least a portion of thebiomass miscible or more functionally compatible, including by reducingthe particle size and/or molecular weight of at least a portion of thebiomass, with the first polymer and the second polymer, (c) contactingthe first polymer with the biomass and the second polymer to form acompound, (d) heating the compound to between 50 degrees Celsius and 250degrees Celsius and/or adding pressure to the compound between about 1and about 5000 atmospheres, and (d) causing the biomass to effect afunctional modification of the first polymer, the second polymer, and/orthe combination of the first polymer and second polymer, wherein thefunctional modification comprises plasticization, nucleation,compatibilization, melt flow modification, increased flexibility,reduced flexular modulus, reduced tensile modulus, increased impactstrength, increased tensile modulus, increased flexular modulus,crystallinity reduction, crystallization rate reduction, increased speedof crystallization, strengthening, and/or elasticization.

In one embodiment, a PHA derived from a carbon-containing greenhousegas, or greenhouse gas emission including methane, carbon dioxide, orcombinations thereof, is provided. In some embodiments, use of such agas is particularly advantageous, as it allows for the simultaneousproduction of PHA at lower energy costs and higher efficiencies, butalso removes a portion of a destructive gas from the atmosphere, orprevents a destructive gas from entering the atmosphere. In someembodiments, processes and systems as disclosed herein are particularlywell suited for use near sources of such gases (e.g., landfills, powerproduction plants, anaerobic digesters, etc.) for onsite conversion ofharmful gasses to a commercially valuable product.

In several embodiments, processes for the oxidation of methane areprovided. In one embodiment, the process comprises: providing a cultureof methanotrophic and autotrophic microorganisms, providing a growthculture medium comprising dissolved methane and carbon dioxide, andcontacting the culture with light to cause the culture to convert thecarbon dioxide into oxygen, whereby the culture utilizes the oxygen tooxidize the methane, thereby reducing or eliminating the need for anextraneous source of oxygen to drive methanotrophic metabolism.

In some embodiments, the light used to contact the culture is artificiallight. In some embodiments, the light used to contact the culture isnatural light. In other embodiments, combinations of natural andartificial light are used. In some such embodiments, wavelengths ofartificial light are specifically filtered out or controlled such thatthe culture is exposed to a broader or more controlled overall spectrumof light (e.g., the sum of wavelengths of natural light and artificiallight). In some embodiments, the source of light also functions togenerate heat, which can be used to maintain optimal culturetemperatures. In other embodiments, light input is regulated by time,such that specific cultures of autotrophic and/or heterotrophicmicroorganisms are selected for or optimized according to the durationand/or pattern of light injection (e.g., 0-12 or 12-24 hours lightinjection, 0-12 or 12-24 hours dark incubation, multi-second pulsation,etc.).

In one embodiment, the addition of light reduces the need for exogenousoxygen sources. While such embodiments provide an advantage in reducingcosts of input materials, in some embodiments, an exogenous source ofoxygen, including air, is added to the culture.

In some embodiments, the addition of autotrophic microorganisms to theculture impacts the metabolism of the culture. In such an embodiment,the timed and planned addition, activation, or metabolic enhancement ofautotrophic organisms can be based on the desire for changing the rateof methane oxidation.

In some embodiments, a system is used for PHA production comprisingproviding i) a culture of autotrophic, methanotrophic, methanogenic,and/or heterotrophic microorganisms and ii) a first gas comprisingcarbon dioxide, methane, volatile organic compounds, oxygen, and/orother gas, whereby the culture of microorganisms are caused to used thefirst gas to generate a second gas comprising carbon dioxide, methane,oxygen, volatile organic compounds, and/or other gas, whereby theculture subsequently is caused to utilize the second gas for thegeneration of PHA, which can then be isolated and purified according toseveral embodiments disclosed herein. In some embodiments, the first gascan be methane, carbon dioxide, oxygen, or volatile organic compounds.In other embodiments, the second gas can be oxygen, methane, carbondioxide, or other volatile organic compounds. In some embodiments,microorganisms can be used to convert carbon dioxide to biomass whichcan in turn be used to produce methane, which can be subsequently usedto produce PHA. In other embodiments, microorganisms can be used toconvert carbon dioxide to oxygen which can in turn be used to producePHA. As disclosed herein, the products generated at each of these stepsmay be recycled (e.g., splitting a portion of the autotrophic cultureand recycling it to generate additional biomass, generating reduced-PHAbiomass and recycling it into the methanotrophic culture to generateadditional biomass and additional PHA).

In several embodiments, processes for producing autotrophicmicroorganisms using only methane as a carbon input are provided. In oneembodiment, the process comprises: adding methane, oxygen, andmethane-utilizing microorganisms to a culture of autotrophicmicroorganisms, whereby the methane-utilizing microorganisms convert themethane into carbon dioxide, and whereby the autotrophic microorganismsutilize the carbon dioxide as a source of carbon, thereby reducing oreliminating the need for an extraneous source of carbon dioxide to driveautotrophic metabolism. In some embodiments, addition of methanotrophicand/or heterotrophic microorganisms to the culture impacts themetabolism of the autotrophic microorganisms. As discussed herein, thepurposeful addition of such microorganisms at particular times allowsfor specific levels of control over the overall output and operation ofthe system.

In several embodiments, processes for oxidizing methane at lowconcentrations are provided. In one embodiment, the process comprises:culturing methanotrophic microorganisms in a medium comprising water,dissolved methane, dissolved oxygen, and mineral salts, adding methanolto the medium at a rate and volume sufficient to cause themicroorganisms to reduce the concentration of the methane in the medium,whereby substantially all of the methane within the medium is utilized,thereby enabling methanotrophic microorganisms to metabolize methanepresent at low bioavailable concentrations. In some embodiments, gascontaining less than 20% methane by volume is contacted with the medium.In some embodiments, gas containing less than 1% methane by volume iscontacted with the medium. In some embodiments, the methanol is producedby microorganism metabolism.

In several embodiments, processes for separating water frommicroorganism biomass are provided. In one embodiment, the processcomprises: providing biomass mixed with water in a liquid medium, mixingthe medium with a liquid agent selected from the group consisting ofketones, alcohols, chlorinated solvents, derivatives thereof, orcombinations thereof, and subjecting the mixture to a filtration step.In several embodiments, this enables the efficient separation of biomassfrom water. In some embodiments, such methods reduce or eliminate theneed for centrifugation in the separation process. In some embodiments,the liquid agent is acetone, ethanol, isopropanol, and/or methanol. Inother embodiments, other liquid agents that are miscible with water areused to separate the biomass from the aqueous portion of the mixture. Inseveral embodiments separation is achieved by centrifugation(high-speed, low-speed), gravity separation, multi-stage filtration, orcombinations thereof.

In several embodiments, processes for extracting a polyhydroxyalkanoatefrom a PHA-containing biomass are provided. In one embodiment, theprocess comprises the steps of: (a) providing a PHA-containing biomasscomprising PHA and water, (b) mixing said biomass with a solvent at atemperature sufficient to dissolve at least a portion of said PHA intosaid solvent and at a pressure sufficient to enable substantially all orpart of said solvent to remain in liquid phase, thereby producing aPHA-lean biomass phase and a PHA-rich solvent phase comprising water,PHA and solvent (c) separating said PHA-rich solvent phase from saidPHA-lean biomass phase at a temperature and pressure sufficient toenable substantially all or part of said solvent to remain in liquidphase and prevent substantially all or part of said PHA within saidPHA-rich solvent phase from precipitating into said water, (d) reducingthe pressure or increasing the temperature of said PHA-rich solventphase to cause said PHA-rich solvent to vaporize and said PHA toprecipitate or otherwise become a solid PHA material while maintainingthe temperature and/or pressure of the PHA-rich solvent phase to preventall or part of the temperature-dependent precipitation of said PHA intosaid water, and (e) collecting said solid PHA material, includingoptionally separating said solid PHA material from said solvent and/orsaid water.

In some embodiments, solvents include acetone, ethanol, methanol,dichloroethane, and/or methylene chloride. Depending on the solventselected, in some embodiments, separating the solid PHA material fromsolvent and/or water is achieved by increasing the temperature of themixture. In other embodiments, separation is achieved through reducingthe pressure of the solvent, PHA, and/or water. In some embodiments,combinations of temperature changes and pressure changes are used tooptimally separate solid PHA material from solvent and/or water. In someembodiments, evaporation of solvent and/or water occurs in a rapidfashion, thereby reducing the need for temperature or pressure changes.Advantageously, certain embodiments of the processes disclosed hereinmay optionally be carried out in a batch, semi-continuous, or continuousmanner. Thus, the process can be tailored to the needs of the producerat any given time.

In several embodiments, processes for modifying the functionalcharacteristics of a PHA are provided. In one embodiment, the processcomprises providing a first PHA and a second PHA, wherein the molecularweight of said second PHA is greater than the molecular weight of saidfirst PHA, and combining said first PHA with said second PHA to modifythe functional characteristics of both said first PHA and second PHA. Insome embodiments, both the first PHA and the second PHA are PHB, and insome embodiments, one or more of the first and second PHA comprisesPHBV. In one embodiment the first PHA and the second PHA is PHB or PHBV.

In some embodiments, the molecular weight of the first PHA is greaterthan about 500,000 Daltons and the molecular weight of the second PHA isless than about 500,000 Daltons. However, in some embodiments, themolecular weight can be adjusted. For example, in some embodiments, afirst PHA is subjected to a temperature sufficient to reduce themolecular weight of the first PHA. Thereafter, it can be combined withthe second PHA. In several embodiments, the second PHA could alsooptionally be exposed to temperature in order to adjust its molecularweight. In some embodiments, the molecular weight of the second PHA isgreater than about 800,000 Daltons. In certain embodiments, themolecular weight of said second PHA is greater than about 1,000,000Daltons. In some embodiments, the molecular weights of the first andsecond PHA are specifically tailored relative to one another, (e.g., aratio of 1:2, 1:4, 1:6, 1:8, 1:10, etc.) in order to maximize thealterations in functional characteristics.

In several embodiments, processes for increasing the penetration depthof light in a liquid are provided. In one embodiment, the processcomprises the steps of (a) directing light into a liquid medium in theform of a light path and (b) reducing the density of liquid in the lightpath.

In several embodiments, the density of the liquid in the light path isreduced by adding gas to said liquid in the light path. In someembodiments, the gas is air, oxygen, methane, carbon dioxide, nitrogen,and/or a combination thereof. In some embodiments, the gas issimultaneously added along with the light. In certain embodiments, thegas and the light are emitted or injected into the liquid through acommon material, such as a permeable or semi-permeable membrane throughwhich light and/or gas traverse. In other embodiments, the light and thegas are added separately. In such embodiments, customization of theaddition is possible. For example, gas can be added in pulses (e.g.,on/off sequences), continuously, or in bracketed time frames around theaddition of light. In some embodiments, the addition of light and gasare coordinated to maximize the penetration of the light. For example aburst of gas followed by a burst of light (or overlapping to somedegree) may advantageously increase the penetration of the light.

In several embodiments, processes for modifying the pH in amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a culture mediumcomprising water and microorganisms, (b) adding a first source ofnitrogen to the medium to cause the microorganisms to metabolize thenitrogen and thereby increase the concentration of either hydroxyl ionsor protons, respectively, in the medium, and/or (c) adding a secondsource of nitrogen to the medium to cause the microorganisms tometabolize the nitrogen and thereby increase the concentration of eitherprotons or hydroxyl ions, respectively, in the medium. In otherembodiments, a source of nitrogen is added to the culture that increasesthe pH of the medium, wherein the metabolism of the nitrogen sourcecauses the pH of the medium to decrease, thereby reducing or eliminatingthe need for an additional pH adjustment step. In one embodiment,nitrogen fixation is used to add hydroxyl ions to the culture medium,which may or may not be counterbalanced by the addition of protons fromeither biological or chemical sources. In one embodiment, nitratefixation is used to add hydroxyl ions to the culture medium, which mayor may not be counterbalanced by the addition of protons from eitherbiological or chemical sources. In one embodiment, ammonia or ammoniumfixation is used to add protons to the culture medium, which may or maynot be counterbalanced by the addition of hydroxyl ions from eitherbiological or chemical sources.

In several embodiments, low shear processes for adding gas to amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a liquid medium and a gas,(b) contacting the medium with the gas in a first container to cause atleast a portion of the gas to dissolve in the medium, (c) providing asecond container, and (d) transferring at least a portion of the liquidcomprising the gas within the first container to the second container.In some embodiments, a mixer is also provided, in order to dissolve aportion of the gas in the medium. In some embodiments, the mixer is apump or agitator or high shear mixer. In some embodiments, the mixercomprises a centrifugal pump. In still other embodiments, the gas itselfprovides a mixing function. For example, the injection of gas (viapreviously existing gas injection methods, including gas injectionmethods which do not require the use of a gas compressor, such as avacuum induction system) into a medium will result in gas bubbles,which, if released at the bottom of a container comprising medium, willnot only promote the dissolution of gas into the medium, but mix themedium as the bubbles rise.

In several embodiments, processes for injecting gas into a pressurizedmicroorganism culture vessel are provided. In one embodiment, theprocess comprises the steps of: providing a vessel comprising a mediumcomprising microorganisms, adding a gas into the vessel that can bemetabolized by the microorganisms, and adjusting the flow rate of thegas into the vessel according to the rate of change of pressure withinthe vessel. In some embodiments, the gas is oxygen, methane, carbondioxide, or combinations thereof. Choice of the gas depends on thevessel used and the culture within the vessel. In some embodiments,backpressure monitoring allows for optimal gas injection for a givenculture (e.g., if certain cultures react more quickly to administrationof a gas and rapidly increase pressure, flow can be coordinatelyreduced).

In one embodiment, a process for producing light in a liquid medium isprovided. In one embodiment, the process comprises the steps of: a)providing a liquid, b) providing a light-emitting unit or materialcomprising two conductive leads and a light-emitting conjuncture betweenthe conductive leads, or a material that will emit light when contactedwith electrons and c) inducing a voltage in the liquid, thereby inducingthe movement of electrons in the conductive leads of the light-emittingunit or inducing electrons to contact the material, thereby causing thelight-emitting unit or material to emit light. In some embodiments, thematerial is a phosphor, phosphoric, and/or luminescent material,including an electroluminescent phosphor.

In one embodiment, AC voltage is induced in a liquid by inserting thetwo leads of a 115V AC power source into a liquid. Without being boundby theory, it is believed that a liquid carrying an AC voltage iscapable of inducing the movement of electrons into and through alight-emitting device suspended in the liquid and not contacting the two115V AC power source leads due to the oscillating nature of electrons inan AC circuit, such that AC voltage in a liquid causes electrons to fillconductive paths connected to the liquid, in spite of the resistance ofthe conductive paths relative to the liquid, and will oscillate as an ACcurrent in those conductive paths, thereby performing work, e.g.,generating light in a light emitting diode. As a non-limiting example,light is produced in a liquid medium by a) placing a light-emittingdiode in a liquid comprising water and electrolytic ions, and b)inducing an AC voltage in the liquid, wherein c) the induction of ACvoltage in the electrolytic liquid causes the light-emitting diode togenerate light.

Through experimentation, Applicant unexpectedly discovered that one ormultiple light emitting units will emit light in a liquid when ACvoltage is applied to the liquid and when the light-emitting units arerated for voltages and amp draws commensurate with available electricalenergy. The production of autotrophic microorganisms is fundamentallyconstrained by the ability of light to penetrate through a liquid andthereby enable photosynthesis. Prior to Applicant's invention asdisclosed herein, no methods were believed to be known to produce lightin a liquid through the utilization of light-emitting devices physicallyunconnected to an electrical voltage source vis-à-vis solid conductivematerial. In one embodiment, the utilization of one or more, andpreferably many, free-floating light-emitting units in an electricallycharged liquid comprising autotrophic microorganisms enables a very highlight transmission efficiency, wherein previous light penetrationconstraints are largely overcome and high autotrophic microorganismdensities are fully enabled.

In several embodiments, a method for producing a polyhydroxyalkanoate(PHA) in a microorganism culture is provided. In some embodiments, themethod comprises the steps of: a) subjecting said culture to a growthperiod comprising exposing said culture to growth conditions to causesaid culture to reproduce, (b) subjecting said culture to apolymerization period comprising exposing said culture to polymerizationconditions to cause said culture to produce intracellular PHA, and (c)repeating step (a) and then second step (b) two or more times.

In some embodiments the growth period comprises a period in which theculture reproduces or otherwise produces biomass and/or reproduces. Insome embodiments, the polymerization period comprises a period in whichthe culture synthesizes PHA. In some embodiments, the growth period andthe polymerization period are induced by the culture media (e.g., theextracellular media around the culture). In some embodiments,alterations in the media conditions induce a transition (partial orcomplete) between growth and polymerization periods. In someembodiments, a culture is cycled between growth and polymerizationperiods two, three, four, or more times, in order to produce PHA andthen reproduce biomass, which is subsequently used to generateadditional PHA.

In some embodiments, the culture is exposed to non-sterile conditions.In certain such embodiments, input carbon is non-sterile. However, insome embodiments, sterile conditions exist. In some embodiments, theculture is dynamic over time in that it may be exposed to extraneousmicroorganisms. In certain embodiments, this is due to a non-sterileculture environment. In some embodiments, extraneous microorganismspotentiate the production of PHA.

In several embodiments, a process for the reduction of pigmentation in amicroorganism culture is provided. In one embodiment, the processcomprises the steps of: providing a medium comprising a microorganismculture comprising dissolved oxygen, and increasing the concentration ofdissolved oxygen over successive periods to select for light-colored orlow-level pigmentation microorganisms.

PHA, while able to be treated post-production to reduce pigmentation, isless expensive to produce when lower levels of pigmentation exist. Somemicroorganisms are more pigmented than others, and therefore in severalembodiments, selection against the more pigmented microorganisms resultsin a less pigmented PHA, which reduces production costs. In someembodiments, the microorganisms cultured and selected for aremethanotrophic microorganisms. By manipulating the culture conditions,which benefit certain varieties of microorganisms, a less pigmentedculture (and hence a less pigmented PHA) result. In some embodiments,increases in concentration of dissolved oxygen over periods ranging from1-3 hours, 3-5 hours, 5-7 hours, 7-10 hours, 10-15 hours, or 15-24 hoursare used to select for less pigmented microorganisms. As discussedherein, in several embodiments, various compounding agents areoptionally added to the produced PHA (either during the production phaseor after production phase). Many such compounding agents, however,adversely affect the quality and/or performance (or othercharacteristic) of the final PHA product. In several embodiments,certain compounding agents, or combinations thereof, are added in orderto improve one or more characteristics of the final PHA product. In someembodiments, such agents aid in the reduction of pigmentation of thePHA. In some embodiments, compounding agents reduce the degree offiltration, centrifugation, settling (or other techniques disclosedherein to separate solids from liquid phases). Advantageously andunexpectedly, the addition of certain compounding agents, viewed in theart as harmful to the quality or to a certain characteristic of the PHA,the result achieve with the methods disclosed herein is a higher qualityPHA that is not adversely affected by the compounding agents, but ratheris enhanced by the addition of such compounds. In several embodiments, aprocess for the conversion of a gas into a polyhydroxyalkanoate (PHA) isprovided, wherein the process comprises the steps of: a) providing i) afirst gas and ii) a culture of microorganisms, b) contacting the firstgas with the culture to cause the culture to convert the first gas intoa second gas c) contacting the second gas with the culture, and d)causing the culture to use the second gas to produce PHA.

In some embodiments, the microorganisms are autotrophic, methanogenic,heterotrophic, or combinations thereof.

In one embodiment the first gas is carbon dioxide. In one embodiment thefirst gas is oxygen. In one embodiment the first gas is methane. In oneembodiment the first gas is a volatile organic compound.

In one embodiment the second gas is carbon dioxide. In one embodimentthe second gas is oxygen. In one embodiment the second gas is methane.In one embodiment the second gas is a volatile organic compound.

In one embodiment, the selection of the first and the second gas isbased on the type of microorganism or microorganisms being cultured.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a block flow diagram comprising the steps of: microorganismfermentation and PHA synthesis, PHA-containing biomass removal,PHA-reduced biomass and isolated PHA production, PHA-reduced biomassrecycling and fermentation, and isolated PHA purification.

DETAILED DESCRIPTION

While PHAs have significant environmental advantages compared to fossilfuel-based plastics, the cost of PHA production is generally viewed as asignificant limitation to the industrial production and commercialadoption of PHAs. Generally, the overall cost of PHA production isdetermined by three major inputs: 1) carbon, 2) chemicals, and 3)energy. Accordingly, efforts to reduce the cost of PHA production mustaddress one or more of these areas, specifically by: i) reducing carboninput costs, ii) increasing carbon-to-PHA yields, iii) reducing thevolume of chemicals required for PHA production, and/or iv) increasingenergy-to-PHA yields.

As discussed above, food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems are widely regarded asthe most carbon, chemical, energy, and, thus, cost efficient PHAproduction method. Despite these efficiencies, sugar-based PHAproduction remains many times more expensive than fossil fuel-basedplastics production. Attempts to reduce the carbon input cost of the PHAproduction process, by utilizing carbon-containing industrial off-gases,such as carbon dioxide and methane, have been previously limited bytechnical challenges and stoichiometric limitations that render thegas-to-PHA production process significantly more energy and chemicalintensive, and thus more costly, than the food crop-based PHA productionprocess.

Specifically, these technical challenges and stoichiometric limitationsinclude: low mass transfer rates, low microorganism growth rates,extended polymerization times, low cell densities, high oxygen demand(relative to solid substrates), and low PHA cellular inclusionconcentrations. Whereas sugar-based fermentation systems have theability to generate high cellular densities and PHA inclusionconcentrations, carbon-containing gas-based fermentation processestypically cannot, based on fundamental cell morphology and mass transferconstraints, generate cellular and PHA densities exceeding 10-30% ofdensities possible in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon injection, oxygeninjection, system cooling, and culture mixing, as well as downstream PHApurification, significantly exceeds the energy-to-PHA ratio required forsugar-based PHA production methods, thereby rendering theemissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based PHAs.

Several embodiments of the present invention therefore relate to a novelmethod for the production of PHA using carbon-containing gases as asource of carbon (alone or in combination with a non-gaseous source ofcarbon), wherein the energy input-to-PHA production ratio, carboninput-to-PHA production ratio, and cost efficiency of the process issignificantly improved over previous gas-based PHA production processes.

In several embodiments, this process may be accomplished by a) culturinga first microorganism culture capable of metabolizing the carbon withinboth a carbon-containing gas and biomass, or a derivative thereof, b)manipulating the conditions of the culture to cause the culture toproduce PHA-containing biomass, c) removing a portion of thePHA-containing biomass; d) extracting at least a portion of the PHAwithin the removed PHA-containing biomass to create substantiallyisolated PHA and substantially PHA-reduced biomass, e) purifying theisolated PHA, and f) returning the PHA-reduced biomass to themicroorganism culture to cause the microorganism culture to metabolizethe carbon within the PHA-reduced biomass into PHA.

According to some embodiments, the steps of this process are as follows:(a) providing a microorganism culture comprising biomass and PHA; (b)removing a portion of the PHA-containing biomass from the culture, andextracting PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass; (c) purifying the isolated PHA,and (d) returning the PHA-reduced biomass to be mixed with the cultureto cause the culture to convert the carbon within the PHA-reducedbiomass into PHA. Each of the above recited steps in the process arediscussed in more detail below.

Providing a Microorganism Culture Comprising Biomass and PHA

The terms “microorganism”, “microorganisms”, “culture”, “cultures”, and“microorganism cultures”, as used herein, shall be given their ordinarymeanings and shall include, but not be limited to, a single strain ofmicroorganism and/or consortium of microorganisms, including, amongothers, genetically-engineered bacteria, fungi, algae, and/or yeast. Insome embodiments, microorganisms are naturally occurring and in someembodiments microorganisms are genetically-engineered. In someembodiments, both naturally occurring and genetically-engineeredmicroorganisms are used. In some embodiments, a mixed culture ofmicroorganisms may be used. In some embodiments, microorganisms orcultures shall include a microorganism metabolism system, including theinteractions and/or multiple functions of multiple cultures in one ormore conditions.

The terms “biomass” and “biomass material” shall be given their ordinarymeaning and shall include, but not be limited to, microorganism-derivedmaterial, including intracellular, cellular, and/or extracellularmaterial, such materials including, but not limited to, a polymer orpolymers, amino acids, nucleic acids, carbohydrates, lipids, sugars,PHA, volatile fatty acids, chemicals, gases, such as carbon dioxide,methane, volatile organic acids, and oxygen, and/or metabolicderivatives, intermediaries, and/or end-products. In severalembodiments, biomass is dried or substantially dried.

In some embodiments, the biomass contains less than about 99% water. Inother embodiments, the biomass contains between about 99% to about 75%water, including about 95%, 90%, 85%, and 80%. In some embodiments, thebiomass contains between about 75% and about 25% water, including75%-65%, 65%-55%, 55%-45%, 45%-35%, 35%-25%, and overlapping rangesthereof. In additional embodiments, the biomass contains from about 25%water to less than about 0.1% water, including 25%-20%, 20%-15%,15%-10%, 10%-5%, 5%-1%, 1%-0.1%, and overlapping ranges thereof. Instill other embodiments, the biomass contains no detectable amount ofwater. Depending on the embodiment, water is removed from the biomass byone or more of freeze drying, spray drying, fluid bed drying, ribbondrying, flocculation, pressing, filtration, and/or centrifugation. Insome embodiments, the biomass may be mixed with one or more chemicals(including, but not limited to solvents), such as methylene chloride,acetone, methanol, and/or ethanol, at various concentrations. In otherembodiments, the biomass may be processed through homogenization, heattreatment, pH treatment, enzyme treatment, solvent treatment, spraydrying, freeze drying, sonication, or microwave treatment. As usedherein, the term “PHA-reduced biomass” shall be given its ordinarymeaning and shall mean any biomass wherein at least a portion of PHA hasbeen removed from the biomass through a PHA extraction process. As usedherein, the term “PHA-containing biomass” shall be given its ordinarymeaning and shall mean any biomass wherein at least a portion of thebiomass is PHA.

Microorganism cultures useful for the invention described herein includea single strain, and/or a consortium of strains, which are individuallyand/or collectively capable of using carbon containing gases andbiomass, including PHA-reduced biomass, as a source of carbon for theproduction of biomass and PHA. In some embodiments, a microorganismculture according to several embodiments, comprises a microorganismculture that utilizes PHA-reduced biomass, or any derivative thereof,including methanotrophic microorganisms, anaerobic digestion cultures,and other heterotrophic microorganisms, as a source of carbon for theproduction of biomass, or metabolic derivatives including, and inparticular, the production of PHA, protein, methane, and/or carbondioxide (herein, “biomass-utilizing microorganisms”). As used herein,the terms “microorganism”, “culture”, “microorganism culture”“microorganism system”, “microorganism consortium”, “microorganismconglomerate” and “consortium of microorganisms” are usedinterchangeably. Additionally, any of these terms may refer to one, two,three, or more microorganism cultures and/or strains, including amicroorganism system that is collectively capable of carrying out acomplex metabolic function (e.g., conversion of PHA-reduced biomass tomethane, carbon dioxide, protein, and/or PHA). In several embodiments,the microorganism culture comprises of a consortium of carbon-containinggas-utilizing microorganisms and a consortium of biomass-utilizingmicroorganisms. In some embodiments, the gases metabolized by suchcultures comprise methane, carbon dioxide, and/or a combination thereof.

In some embodiments, the microorganism culture comprises a consortium ofacidogenic, acetogenic, methanogenic, methanotrophic, and/or autotrophicmicroorganisms in one or more individual bioreactors. As such, in someembodiments, the cultures are grown in one or more distinct cultureconditions. In some embodiments, the conditions are either aerobic oranaerobic conditions. In some embodiments, culture conditions are variedover time (e.g. initially aerobic with a transition to anaerobic, orvice versa). As used herein, the term “bioreactor” shall be given itsordinary meaning and shall also refer to a tank, vessel, group ofvessels, tank of vessels, or any device or system suitable for growthand culturing of microorganisms. In one embodiment, a device is providedthat is capable of carrying out gas-based fermentation, methanotrophicmetabolism, bioreaction, autotrophic metabolism, heterotrophicmetabolism, and/or biocatalyst-based metabolism at high efficiency,particularly using one or more, and particularly at least two gases asnutrient (e.g., carbon and oxygen) input sources, measured in thefollowing terms: 1) gas capture efficiency, 2) mass transfer efficiency(including in terms of the power required to transfer gas intoaqueous/dissolved form), and 3) material synthesis (in terms of gramsper liter per hour). In one embodiment, a system is provided for gasinput reactions (e.g., methane and oxygen; oxygen and carbon dioxide;carbon dioxide and methane; methane, ammonia, and oxygen; methane,ammonia, oxygen, and dinitrogen; methane, carbon dioxide, and oxygen; orvarious combinations of such input gasses) that utilizes a systemcomprising multiple reaction vessels. In one embodiment, one or morevessel may be equipped with a rotating mixer. In one embodiment, therotating mixer may induce cavitation in the liquid medium. In oneembodiment, such cavitation may cause acute induction of gas entrainmentinto the liquid medium, significantly increasing mass transferinduction. In one embodiment, gas may be injected into one or more ofthe vessels behind the leading edge of a moving material in liquidmedium, in order to reduce and then increase the driving pressure of thegas injection. In one embodiment, the pressure of the liquid medium maybe pulsed through periods of high pressure and low pressure to increasethe mass transfer of gas into liquid medium. In one embodiment, thepulsation of pressure in a liquid medium may be employed, wherein thehigh pressure (e.g., up to 100 psi) period may have a duration from0.001 seconds to 25 minutes, and wherein the low pressure period (e.g.,from −25 inches vacuum to 5 psi) may have a duration from 0.001 secondsto 25 minutes. In one embodiment, the rapid induction of pressurepulsation may be effected by fitting a vessel with a means oftransferring acoustic energy into the vessel medium. In one embodiment,the rapid induction of pressure pulsation may be effected by fitting avessel with a transducer. In one embodiment, the rapid induction ofpressure pulsation may be effected by fitting a vessel with one or moresonication means, wherein the liquid medium is sonicated, wherein suchsonication is diffused throughout a volume sufficient to avoid damage tomicroorganisms or enzymes in the liquid medium. In one embodiment,silica gel or silica-based liquid is added to the liquid medium toincrease the solubility of methane and oxygen in the liquid medium. Inone embodiment, the reaction vessels comprise fully or partiallyenclosed vessels. In one embodiment, the reaction vessels comprise fullyor partially-enclosed medium-containing volumes or medium-containingcompartments, within or in addition to one or more tanks, compartments,vessels, or other volumes. In one embodiment, the vessels may be plasticor stainless steel enclosed vessels or medium-containing volumes. In oneembodiment, the vessels may not be physically connected. In oneembodiment, the vessels may be physically connected. In one embodiment,gas may be directed into one or more of the vessels simultaneously. Inone embodiment, a reactor, reactor system, or system may comprisemultiple vessels combined. In one embodiment, gas may be directedequally into each of vessels. In one embodiment, gas may be directedmore into one vessel and less into another vessel. In one embodiment,gas may be directed first into one vessel, and then into another vessel.In one embodiment, gas may be exhausted from all vessels equally. In oneembodiment, gas may be exhausted from all vessels individually, or morefrom one vessel and less from another vessel. In one embodiment, exhaustgas may be directed from one vessel into another vessel. In oneembodiment, the liquid medium of the vessels is discrete and not mixedbetween the vessels. In one embodiment, the liquid medium of the vesselsis not discrete and is mixed between the vessels. In one embodiment, gasis directed equally into all vessels, and liquid medium is mixed betweenthe vessels. In one embodiment, gas is directed equally into allvessels, and liquid medium is at least partially mixed between thevessels. In one embodiment, gas is directed equally into all vessels,and liquid medium is at least partially mixed between the vessels. Inone embodiment, gas is directed equally into all vessels, and liquidmedium is not mixed between the vessels. In one embodiment, gas isdirected first into one vessel and then into another vessel, and liquidmedium is mixed between the vessels. In one embodiment, gas is directedfirst into one vessel and then into another vessel, and liquid medium isnot mixed between the vessels. In one embodiment, exhaust gas from afirst vessel is directed into a second vessel, and liquid medium ismixed between the vessels. In one embodiment, gas is directedindividually and discretely into each vessel, and liquid medium is mixedbetween the vessels. In one embodiment, exhaust gas is directedindividually and discretely into each vessel, and liquid medium is notmixed between the vessels. In one embodiment, the concentration ofdissolved gas is caused to remain relatively elevated in one vessel andrelatively depressed in another vessel. In one embodiment, theconcentration of dissolved gas is caused to remain substantially equalin multiple vessels. In one embodiment, the gases are caused to be mixedequally throughout the vessels. In one embodiment, the gases are causedto move sequentially through the vessels. In one embodiment, the gasesare caused to be injected individually into through the vessels. In oneembodiment, the gases are caused to be injected individually andsimultaneously into the vessels. In one embodiment, the gases are causedto move simultaneously through the vessels on an individual basis andmedium is caused to not be fully mixed between the vessels, such thatthe medium remains substantially isolated. In one embodiment, the gasesare caused to move sequentially through the vessels and medium is causedto not be fully mixed between the vessels. In one embodiment, the gasesare caused to be injected individually and simultaneously into thevessels and medium is caused to be mixed between the vessels, such thatthe medium remains substantially non-isolated. In one embodiment, thegases are caused to move sequentially through the vessels to cause themedium in the vessels to be substantially non-isolated and gas is causedto move in multiple directions in each vessel. In one embodiment, thegases are caused to be injected simultaneously into the vessels, suchthat the gases are caused to move the medium in the vessels to besubstantially non-isolated. In one embodiment, the gases are caused tobe injected simultaneously into the vessels in such a manner that thegases are not caused to move the medium between the vessels. In oneembodiment, gas may be moved between vessels by mechanical means, suchas a pump. In one embodiment, liquid medium may be moved between vesselsby mechanical means, such as a pump. In one embodiment, gas may beinjected into a vessel by mechanical means. In one embodiment, liquidmedium may be injected into a vessels by mechanical means, such as apump. In one embodiment, gas and liquid medium may be injectedsimultaneously into a vessel by mechanical means, such as a pump,nozzle, venturi, compressor, diffusor, vacuum. In one embodiment, thevessels may be equipped with one or more internal cavitation mechanisms.In one embodiment, the vessels may be operated under recurring periodsor patterns of pressure and vacuum to induce optimal mass transferefficiency. In one embodiment, the vessels may be filled with one ormore materials, that are more or less dense than liquid medium, that areable to dissolve or absorb high concentrations or amounts of gases, suchas methane, oxygen, or carbon dioxide, wherein such materials may besilica-based gels or beads, activated carbon, nickel-plated spheres,polypropylene beads, PES beads, PTFE beads, or ultra high molecularweight polyethylene pellets. In one embodiment, the rapid pulsation ofpressure in the vessels causes the absorbent materials to absorb gasesat high concentration, and then release at least some of the gases intothe medium, causing an increase in mass transfer into the liquid medium.In one embodiment, a vessel is filled with liquid medium containinggas-absorbent material, such as plastic beads, and the vessel is subjectto recurring periods of pressurization, such that the vessel actssimilar to an oxygen concentration system or other pressure swingabsorption system, thereby increasing the solubility, mass transfer,and/or uptake of gases in the vessel by the microorganisms and liquidmedium. In one embodiment, such pressure or depressurization cycle maycomprise 1-100 minutes per cycle or stage, or 1-300 minutes per completepressure-depressurization cycle. In one embodiment, the liquid mediumand/or concentration of dissolved gases in the reactor is caused toremain relatively constant or homogenous with mixing induced by theaction of the cavitation (e.g., cavitation induced by a moving blade orliquid moving over a surface), sonication (e.g., ultrasonication), sonicinduction, gases (e.g., gas displacement), liquid displacement,mechanical pumping (e.g., rotary pump), the movement of entrainedmaterials (e.g., the movement of liquid-entrained plastic balls), orother means while the concentration of gases is caused to be reduced ona proximal basis according to the cycle of pressure in the system (e.g.,from vacuum pressure to superatmospheric pressure), proximity to anabsorbent material (including the associated pressure cycle), sequentiallocation of gas relative to gas flow path (e.g., location in gasvessels), and location or proximity to injection port relative toexhaust port (e.g., retention time of gas). In one embodiment, a reactormay be vertically configured, such that the height of the vessel exceedsthe width of the vessel. In one embodiment, a reactor may behorizontally configured, such that the width of the vessel exceeds theheight of the vessel.

In some embodiments, the microorganism culture is contained within asingle vessel, wherein the steps of converting PHA-reduced biomass tobiomass, converting biomass to PHA, and converting carbon-containinggases to biomass and/or PHA occur simultaneously or sequentially.

In other embodiments, the microorganism culture is contained withinmultiple vessels, which are designed to carry out specific and uniquefunctions. For example, one embodiment includes the steps of (a)converting PHA-reduced biomass to PHA-reduced biomass-derived materialssuch volatile organic acids, methane, and/or carbon dioxide, which iscarried out in a first vessel and (b) synthesizing PHA from PHA-reducedbiomass-derived materials which is carried out in a second, separatetank under independent conditions. In some embodiments, one or more ofthe tanks is an anaerobic digestion tank and one or more other tank isan aerobic fermentation tank.

As used herein, the term “gas-utilizing microorganisms” shall be givenits ordinary meaning and shall refer to microorganisms capable ofutilizing gases containing carbon for the production of biomass,including the production of PHA. Similarly, the terms “methanotrophicmicroorganisms” and “methane-utilizing microorganisms” shall be giventheir ordinary meanings and shall refer to microorganisms capable ofutilizing methane as a source of carbon for the production of biomass.Further, the terms “autotrophic microorganisms” and “carbondioxide-utilizing microorganisms” shall be given their ordinary meaningand shall refer to microorganisms capable of utilizing carbon dioxide asa source of carbon for the production of biomass, includingmicroorganisms that utilize natural and/or synthetic sources of light tocarry out the metabolism of carbon dioxide into biomass. The term“heterotrophic microorganisms”, as used herein, shall be given itsordinary meaning and shall include methanotrophic, methanogenic,acidogenic, acetogenic and biomass-utilizing microorganisms, includingmicroorganisms that convert sugar, volatile fatty acids, or other carbonsubstrates to biomass. The term “methanogenic microorganisms” shall begiven its ordinary meaning and shall refer to microorganisms thatconvert biomass to methane, including the consortium of microorganismsrequired to carry out such a process, including, but not limited to,acidogenic and acetogenic microorganisms.

As discussed herein, in several embodiments, carbon-containing gases areused as a source of carbon by microorganism cultures. In someembodiments, other sources of carbon are used (e.g., PHA-reducedbiomass), either alone or in combination with carbon-containing gases.In some embodiments, the carbon-containing gases used include, but arenot limited to, carbon dioxide, methane, ethane, butane, propane,benzene, xylene, acetone, methylene chloride, chloroform, volatileorganic compounds, hydrocarbons, and/or combinations thereof. The sourceof the carbon-containing gases depends on the embodiment. For example,carbon-containing gas sources used in some embodiments includelandfills, wastewater treatment plants, anaerobic metabolism, powerproduction facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, cement production facilities, and/oranaerobic organic material digesters, including both solid and liquidmaterial digesters.

In several embodiments described herein, microorganisms may include, butare not limited to, yeast, fungi, algae, and bacteria (includingcombinations thereof). Suitable yeasts include, but are not limited to,species from the genera Candida, Hansenula, Torulopsis, Saccharomyces,Pichia, 1-Debaryomyces, Lipomyces, Cryptococcus, Nematospora, andBrettanomyces. Suitable genera include Candida, Hansenula, Torulopsis,Pichia, and Saccharomyces. Non-limiting examples of suitable speciesinclude, but are not limited to: Candida boidinii, Candida mycoderma,Candida utilis, Candida stellatoidea, Candida robusta, Candidaclaussenii, Candida rugosa, Brettanomyces petrophilium, Hansenulaminuta, Hansenula satumus, Hansenula californica, Hansenula mrakii,Hansenula silvicola, Hansenula polymorpha, Hansenula wickerhamii,Hansenula capsulata, Hansenula glucozyma, Hansenula henricii, Hansenulanonfermentans, Hansenula philodendra, Torulopsis candida, Torulopsisbolmii, Torulopsis versatilis, Torulopsis glabrata, Torulopsismolishiana, Torulopsis nemodendra, Torulopsis nitratophila, Torulopsispinus, Pichia farinosa, Pichia polymorpha, Pichia membranaefaciens,Pichia pinus, Pichia pastoris, Pichia trehalophila, Saccharomycescerevisiae, Saccharomyces fragilis, Saccharomyces rosei, Saccharomycesacidifaciens, Saccharomyces elegans, Saccharomyces rouxii, Saccharomyceslactis, and/or Saccharomyces fractum.

In several embodiments, mutants (including genetically-engineered ornaturally occurring) of the above-referenced yeasts are used. Forexample, in several embodiments, mutants having about 99.9%, about 99%,about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about92%, about 91%, about 90%, about 85%, about 80%, about 70%, about 60%,about 50%, about 25%, or about 10% genetic homology (e.g., in comparinggenome to genome) to the above-referenced yeasts are used. In someembodiments, microorganisms are used in which particular genes(including groups of genes or families of genes) are mutated such thatone or more of the genes exhibit less than 100% sequence similarity(e.g., about 99.9%, about 99%, about 98%, about 97%, about 96%, about95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%,about 80%, about 70%, about 60%, about 50%, about 25%, or about 10%sequence homology) to a corresponding gene (or genes) in themicroorganisms disclosed herein. In some embodiments, one or more pointmutations in the DNA of the microorganism account for the mutant status.In some embodiments, the point mutation(s) are transitions, in someembodiments they are transversions, and in some embodiments wherein morethan one point mutation is present, combinations of transitions andtransversions exist. In some embodiments, the mutations are nonsensemutations, missense mutations, silent mutations, or combinationsthereof. In some embodiments, the mutations are deletions, insertions orcombinations thereof. In some embodiments, such mutations lead toframeshifts in the genetic code, which may lead to alterations in theresultant protein. In some embodiments, the genetic discrepancies resultin altered protein (e.g., non-functional, reduced function, truncated,non-expressed) as compared to a non-mutant bacteria. In someembodiments, non-functional or reduced function proteins exhibit lessthan about 99%, about 95%, about 90%, about 85%, about 80%, about 70%,about 60%, about 50%, about 25%, or about 10% of the activity of anormal protein. In some embodiments, truncated proteins are partiallyfunctional, while in some embodiments, they are non-functional. In someembodiments, the proteins are not expressed. In some embodiments,post-translational modification of proteins results in alteredexpression or function of one or more proteins of the microorganism(e.g., an enzyme in a metabolic pathway). Such modifications include,but are not limited to, myristoylation, palmitoylation, isoprenylationor prenylation, farnesylation, geranylgeranylation, glypiation,glycosylphosphatidylinositol anchor formation, lipoylation, flavination,heme C attachment, phosphopantetheinylation, retinylidene Schiff baseformation, diphthamide formation, ethanolamine phosphoglycerolattachment, hypusine formation, acylation, N-acylation (amides),S-acylation (thioesters), acetylation, deacetylation, formylation,alkylation, methylation, demethylation, amide bond formation, amidationat C-terminus, amino acid addition (e.g., arginylation,polyglutamylation, polyglycylation), butyrylation, gamma-carboxylation,glycosylation, glycation, polysialylation, malonylation, hydroxylation,iodination, nucleotide addition such as ADP-ribosylation, oxidation,phosphate ester (O-linked) or phosphoramidate (N-linked) formation,phosphorylation, adenylylation, propionylation, pyroglutamate formation,S-glutathionylation, S-nitrosylation, succinylation, sulfation,selenoylation, glycation, biotinylation, pegylation, ISGylation,SUMOylation, ubiquitination, Neddylation, Pupylation, citrullination,deimination, deamidation, eliminylation, carbamylation, formation ofdisulfide bridges, proteolytic cleavage, and racemization.

Suitable bacteria include, but are not limited to, species from thegenera Bacillus, Mycobacterium, Actinomyces, Nocardia, Pseudomonas,Methanomonas, Protaminobacter, Methylococcus, Arthrobacter,Methylomonas, Brevibacterium, Acetobacter, Methylomonas, Brevibacterium,Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium,Rhodopseudomonas, Microbacterium, Achromobacter, Methylobacter,Methylosinus, and Methylocystis. Preferred genera include Bacillus,Pseudomonas, Protaminobacter, Micrococcus, Arthrobacter and/orCorynebacterium. Non-limiting examples of suitable species include, butare not limited to: Bacillus subtilus, Bacillus cereus, Bacillus aureus,Bacillus acidi, Bacillus urici, Bacillus coagulans, Bacillus mycoides,Bacillus circulans, Bacillus megaterium, Bacillus licheniformis,Pseudomonas ligustri, Pseudomonas orvilla, Pseudomonas methanica,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas oleovorans,Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas pyocyanea,Pseudomonas methylphilus, Pseudomonas brevis, Pseudomonas acidovorans,Pseudomonas methanoloxidans, Pseudomonas aerogenes, Protaminobacterruber, Corynebacterium simplex, Corynebacterium hydrocarbooxydans,Corynebacterium alkanum, Corynebacterium oleophilus, Corynebacteriumhydrocarboclastus, Corynebacterium glutamicum, Corynebacterium viscosus,Corynebacterium dioxydans, Corynebacterium alkanum, Micrococcuscerificans, Micrococcus rhodius, Arthrobacter rufescens, Arthrobacterparafficum, Arthrobacter citreus, Methanomonas methanica, Methanomonasmethanooxidans, Methylomonas agile, Methylomonas albus, Methylomonasrubrum, Methylomonas methanolica, Mycobacterium rhodochrous,Mycobacterium phlei, Mycobacterium brevicale, Nocardia salmonicolor,Nocardia minimus, Nocardia corallina, Nocardia butanica,Rhodopseudomonas capsulatus, Microbacterium ammoniaphilum,Archromobacter coagulans, Brevibacterium butanicum, Brevibacteriumroseum, Brevibacterium flavum, Brevibacterium lactofermentum,Brevibacterium paraffinolyticum, Brevibacterium ketoglutamicum, and/orBrevibacterium insectiphilium.

In several embodiments, mutants (including genetically-engineered ornaturally occurring) of the above-referenced bacteria are used. Forexample, in several embodiments, mutants having about 99.9%, about 99%,about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about92%, about 91%, about 90%, about 85%, about 80%, about 70%, about 60%,about 50%, about 25%, or about 10% genetic homology (e.g., in comparinggenome to genome) to the above-referenced bacteria are used. In someembodiments, microorganisms are used in which particular genes(including groups of genes or families of genes) are mutated such thatone or more of the genes exhibit less than 100% sequence similarity(e.g., about 99.9%, about 99%, about 98%, about 97%, about 96%, about95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 85%,about 80%, about 70%, about 60%, about 50%, about 25%, or about 10%sequence homology) to a corresponding gene (or genes) in the bacteriadisclosed herein. In some embodiments, one or more point mutations inthe DNA of the microorganism account for the mutant status. In someembodiments, the point mutation(s) are transitions, in some embodimentsthey are transversions, and in some embodiments wherein more than onepoint mutation is present, combinations of transitions and transversionsexist. In some embodiments, the mutations are nonsense mutations,missense mutations, silent mutations, or combinations thereof. In someembodiments, the mutations are deletions, insertions or combinationsthereof. In some embodiments, such mutations lead to frameshifts in thegenetic code, which may lead to alterations in the resultant protein. Insome embodiments, the genetic discrepancies result in altered protein(e.g., non-functional, reduced function, truncated, non-expressed) ascompared to a non-mutant bacteria. In some embodiments, non-functionalor reduced function proteins exhibit less than about 99%, about 95%,about 90%, about 85%, about 80%, about 70%, about 60%, about 50%, about25%, or about 10% of the activity of a normal protein. In someembodiments, truncated proteins are partially functional, while in someembodiments, they are non-functional. In some embodiments, the proteinsare not expressed. In some embodiments, post-translational modificationof proteins results in altered expression or function of one or moreproteins of the microorganism (e.g., an enzyme in a metabolic pathway).Such modifications include, but are not limited to, myristoylation,palmitoylation, isoprenylation or prenylation, farnesylation,geranylgeranylation, glypiation, glycosylphosphatidylinositol anchorformation, lipoylation, flavination, heme C attachment,phosphopantetheinylation, retinylidene Schiff base formation,diphthamide formation, ethanolamine phosphoglycerol attachment, hypusineformation, acylation, N-acylation (amides), S-acylation (thioesters),acetylation, deacetylation, formylation, alkylation, methylation,demethylation, amide bond formation, amidation at C-terminus, amino acidaddition (e.g., arginylation, polyglutamylation, polyglycylation),butyrylation, gamma-carboxylation, glycosylation, glycation,polysialylation, malonylation, hydroxylation, iodination, nucleotideaddition such as ADP-ribosylation, oxidation, phosphate ester (0-linked)or phosphoramidate (N-linked) formation, phosphorylation, adenylylation,propionylation, pyroglutamate formation, S-glutathionylation,S-nitrosylation, succinylation, sulfation, selenoylation, glycation,biotinylation, pegylation, ISGylation, SUMOylation, ubiquitination,Neddylation, Pupylation, citrullination, deimination, deamidation,eliminylation, carbamylation, formation of disulfide bridges,proteolytic cleavage, and racemization.

In several embodiments, more than one type or species of microorganismis used. For example, in some embodiments, both algae and bacteria areused. In some embodiments, several species of yeast, algae, fungi,and/or bacteria are used. In some embodiments, a single yeast, algae,fungi, and/or bacteria species is used. In some embodiments, aconsortium of cyanobacteria is used. In some embodiments, a consortiumof methanotrophic microorganisms is used. In still additionalembodiments, a consortium of both methanotrophic bacteria andcyanobacteria are used. In several embodiments, methanotrophic,heterotrophic, methanogenic, and/or autotrophic microorganisms are used.

In several embodiments of the invention, the microorganism culturecomprises a consortium of methanotrophic, autotrophic, and/orheterotrophic microorganisms, wherein methane and/or carbon dioxide isindividually, interchangeably, or simultaneously utilized for theproduction of biomass. In some embodiments, PHA-reduced biomass is usedas a source of carbon by heterotrophic, autotrophic, and/ormethanotrophic microorganisms. In several embodiments of the invention,the microorganism culture comprises methanotrophic microorganisms,cyanobacteria, and non-methanotrophic heterotrophic microorganisms,wherein methane and carbon dioxide are continuously utilized as sourcesof carbon for the production of biomass and PHA.

In some embodiments, microorganisms are employed in a non-sterile, open,and/or mixed environment. In other embodiments, microorganisms areemployed in a sterile and/or controlled environment.

The terms “PHA”, “PHAs”, and “polyhydroxyalkanoate”, as used herein,shall be given their ordinary meaning and shall include, but not belimited to, polymers generated by microorganisms as energy and/or carbonstorage vehicles; biodegradable and biocompatible polymers that can beused as alternatives to petrochemical-based plastics such aspolypropylene, polyethylene, and polystyrene; polymers produced bybacterial fermentation of sugars, lipids, or gases; and/or thermoplasticor elastomeric materials derived from microorganisms. PHAs include, butare not limited to, polyhydroxybutyrate (PHB), polyhydroxyvalerate(PHV), polyhydroxybutyrate-covalerate (PHBV), and polyhydroxyhexanoate(PHHx), as well as both short chain length (SCL), medium chain length(MCL), and long chain length (LCL) PHAs.

The terms “growth-culture medium”, “growth medium”, “growth-culturemedia”, “medium”, and “media”, as used herein shall be given theirordinary meaning and shall also refer to materials affecting the growth,metabolism, PHA synthesis, and/or reproductive activities ofmicroorganisms. Non-limiting examples of growth-culture media used inseveral embodiments include a mineral salts medium, which may comprisewater, nitrogen, vitamins, iron, phosphorus, magnesium, and variousother nutrients suitable to effect, support, alter, modify, control,constrain, and/or otherwise influence the metabolism and metabolicorientation of microorganisms. A growth-culture medium may comprisewater filled with a range of mineral salts. For example, each liter of aliquid growth-culture medium may be comprised of about 0.7-1.5 g KH₂PO₄,0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃, 0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28mg CaCl₂*2H₂O, 5.0-5.4 mg EDTA Na₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O,0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mg MnCl₂*2H₂O, 0.06-0.08 mg ZnCl₂,0.05-0.07 mg H₃BO₃, 0.023-0.027 mg NiCl₂*6H₂O, 0.023-0.027 mgNaMoO₄*2H₂O, and 0.011-0.019 mg CuCl₂*2H₂O. A growth-culture medium canbe of any form, including a liquid, semi-liquid, gelatinous, gaseous,foam, or solid substrate.

In several embodiments of the invention, a microorganism culture isproduced in a liquid growth medium, wherein carbon dioxide and methaneare utilized as a gaseous source of carbon for the production ofmethanotrophic and/or autotrophic biomass. In some embodiments,PHA-reduced methanotrophic and/or PHA-reduced autotrophic biomass isutilized as a source of carbon for the production of heterotrophicbiomass and heterotrophically-produced PHA. In some embodiments, thegrowth medium is manipulated to effect the growth, reproduction, and PHAsynthesis of the microorganism culture. Methods for the production ofmethanotrophic microorganisms are disclosed in the art, and aredescribed by Herrema, et al., in U.S. Pat. No. 7,579,176, which ishereby incorporated by reference in its entirety. Methods for theproduction of cyanobacteria are described by Lee, et al. (“High-densityalgal photobioreactors using light-emitting diodes,” Biotechnology andBioengineering, Vol. 44, Issue 10, pp. 1161-1167), which is herebyincorporated by reference in its entirety. Methods for the production ofmethane from biomass are described by Deublein, et al. (“Biogas fromWaste and Renewable Resources, WILEY-VCH Verlag GmbH & Co. KgaA, 2008),which is hereby incorporated by reference in its entirety. In someembodiments, PHA synthesis may be effected through the manipulation ofone of more elements of the culture medium, including through thereduction, increase, or relative change in either the total orbioavailable concentration of one or more of the following elements(also referred to as essential nutrients): carbon, oxygen, magnesium,phosphorus, phosphate, potassium, sulfate, sulfur, calcium, boron,aluminum, chromium, cobalt, iron, copper, nickel, manganese, molybdenum,sodium, nitrogen, nitrate, ammonia, ammonium, urea, amino acids,methane, carbon dioxide, and/or hydrogen. Methods for the production ofPHA are described by Herrema, et al., in U.S. Pat. No. 7,579,176.Depending on the embodiment, all of the various components (includingelements, compounds, liquids, gases, solids, and other compositions) ofa culture medium can be considered essential nutrients, given that theysupport the growth of the microorganisms.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is effected by manipulating the concentration one or more elementswithin a growth medium selected from the group consisting of: nitrogen,methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium, iron,copper, sulfate, manganese, calcium, chlorine, boron, zinc, aluminum,nickel, and/or sodium, and combinations thereof. Methods to control theconcentration of elements within the medium include, but are not limitedto, automatic, continuous, batch, semi-batch, manual, injection, solidfeed, liquid, or other methods of inputting one or more chemical intothe medium, wherein the total and/or bioavailable concentration ofelements is increased, decreased, maintained, adjusted, or otherwisecontrolled at one or more time and/or physical chemical adjustmentpoints. Additional methods to adjust the total or bioavailableconcentration of one or more elements within a mineral media include,but are not limited to, the directed precipitation, chelation,de-chelation, and de-precipitation of elements. In one embodiment, thedirected precipitation or chelation of one or more element is utilizedto reduce the total or bioavailable concentration of one or more elementwithin a medium and thereby induce or increase PHA production in abiomass. In one embodiment, an ion exchange system, including one ormore reversible ion exchange resins, is used to control theconcentration of ions with the medium and/or control the precipitationor solubilization of elements within the medium. In one embodiment, themedium is passed through an ion exchange resin in order to induce thereduction or increase of a specific ion in the medium in order to induceor preclude PHA production in a biomass.

In one embodiment, the concentration of one or more elements within agrowth culture medium is increased, controlled, manipulated, or managedto induce or increase the rate of PHA production in biomass, including amicroorganism culture. As used herein, the terms “control”,“manipulate”, “adjust”, “maintain”, “manage” and the like shall be giventheir ordinary meaning and shall also refer to steps which are taken tokeep or achieve concentrations of certain nutrients, compounds orelements of a culture within a desired range. For example, in onecontext controlling the concentration of an element may result in themaintenance of the concentration of that element within a certain range,that concentration being achieved by the addition of that element and/ordilution of the culture to reduce the concentration of that element. Inone embodiment, the concentration of phosphorus within the medium isincreased to induce or increase the rate of PHA production in amicroorganism culture. In another embodiment, the concentration of anelement, e.g., phosphorus, carbon dioxide, iron, copper, oxygen,methane, and/or magnesium, within the medium is manipulated or increasedto cause a metabolic shift in the microorganism culture, such that theproduction of non-PHA materials by the culture using nitrogen sources(e.g., nitrate, ammonia, ammonium, dinitrogen, urea, or amino acids) isreduced, inhibited, or otherwise impacted to enhance PHA production. Inone embodiment, the concentration of phosphorus within the medium ismanipulated or increased to reduce the utilization of nutrients,including nitrogen, oxygen, and/or carbon, for the production of non-PHAmaterials by the culture. In another embodiment, the concentration ofphosphorus within the medium is manipulated or increased to reduce theutilization of nutrients for the production of non-PHA materials by theculture and induce or increase the rate of PHA production in theculture. In some embodiments, an increase in the concentration ofphosphorus causes a metabolic shift that favors the production of PHA atthe expense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars, lipids,particularly but not necessarily under growth-limiting conditions,including nitrogen (e.g., nitrate, ammonia, ammonium, dinitrogen, urea,or amino acids), oxygen, magnesium, potassium, iron, copper, or othernutrient-limiting conditions. In some embodiments, an increase in theconcentration of phosphorus above 0.00 ppm, 0.01 ppm, 0.02 ppm, 0.05ppm, 0.10 ppm, 0.20 ppm, 0.50 ppm, 1.00 ppm, 1.25 ppm, 1.50 ppm, 1.75ppm, 2.00 ppm, 2.20 ppm, 2.40 ppm, 3 ppm, 4 ppm 5 ppm, 6 ppm, 8 ppm, 20mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or120 mM, and particularly above 40 mM or 80 mM, causes a reduction in theutilization of carbon and nitrogen sources for the production of non-PHAmaterial, including proteins, non-PHA polymers, nucleic acids, lipids,pigments, polysaccharides, methanobactin, and/or carbon dioxide, and,under growth-limiting conditions, an increase in the utilization ofcarbon sources for the production of PHA material, as a result ofmetabolic changes in the culture and/or chemical interactions betweenchemicals within the media and/or culture induced by augmentedconcentrations of phosphorus. The elevation of phosphorus concentrationsas a method to induce or increase the rate of PHA production in abiomass culture is contrary to the teachings of the prior art, whichteaches that PHA production is induced or enhanced by reducing oreliminating the concentration of elements, such as, e.g., phosphorus inthe mineral medium. The addition or controlled elevation of an element,such as, e.g., phosphorus to a biomass system to induce or increase PHAproduction produces an unexpected and surprising increase in PHAproduction in a biomass system. An element such as, e.g., phosphorus maybe added to the mineral media using a variety of phosphorus sources,including phosphorus, phosphate, phosphoric acid, sodium phosphate,disodium phosphate, monosodium phosphate, and/or potassium phosphate;other elements, such as dissolved carbon dioxide, Fe(II) iron, Fe(III)iron, copper sulfate, Fe-EDTA, dissolved oxygen, dissolved methane,and/or magnesium sulfate, hydrogen sulfide, and sodium hydroxide areamong other potential sources of elements that may be added to themineral media.

In one embodiment of the invention, the concentrations of dissolvedgases, such as methane, oxygen, carbon dioxide, and/or nitrogen, aremanipulated to increase the rate of PHA production relative to the rateof cellular production of non-PHA materials and, specifically, to causea reduction in the utilization of carbon or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,enzymes, nucleic acids, lipids, pigments, polysaccharides, and/or carbondioxide, and, under some conditions, including growth-limitingconditions, further cause an increase in the utilization of carbonsources for the production of PHA material, as a result of metabolicchanges in the culture and/or chemical interactions between chemicalswithin the media and/or culture induced by augmented concentrations ofone or more of such dissolved gases. In one embodiment, theconcentration of methane or dissolved methane is manipulated to betweenabout 0.01 ppm and about 0.05 ppm, between about 0.05 ppm and about 0.1ppm, between about 0.1 ppm and about 0.5 ppm, between about 0.5 ppm andabout 1.0 ppm, between about 1.0 ppm and about 1.5 ppm, between about1.5 ppm and about 1.75 ppm, between about 1.75 ppm and about 2.0 ppm,between about 2.0 ppm and about 2.5 ppm, between about 2.5 ppm and about3.0 ppm, between about 3.0 ppm and about 3.5 ppm, between about 3.5 ppmand about 4.0 ppm, between about 4.0 ppm and about 4.5 ppm, betweenabout 4.5 ppm and about 5.0 ppm, between about 5.0 ppm and about 6.0ppm, between about 6.0 ppm and about 7.0 ppm, between about 7.0 ppm andabout 8.0 ppm, between about 8.0 ppm and about 10 ppm, between about 10ppm and about 15 ppm, between about 15 ppm and about 20 ppm, betweenabout 20 ppm and about 30 ppm, or between about 30 ppm and about 50 ppm(and overlapping ranges thereof) to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inone embodiment, the concentration of oxygen or dissolved oxygen ismanipulated to between about 0.01 ppm and about 0.05 ppm, between about0.05 ppm and about 0.1 ppm, between about 0.1 ppm and about 0.5 ppm,between about 0.5 ppm and about 1.0 ppm, between about 1.0 ppm and about1.5 ppm, between about 1.5 ppm and about 1.75 ppm, between about 1.75ppm and about 2.0 ppm, between about 2.0 ppm and about 2.5 ppm, betweenabout 2.5 ppm and about 3.0 ppm, between about 3.0 ppm and about 3.5ppm, between about 3.5 ppm and about 4.0 ppm, between about 4.0 ppm andabout 4.5 ppm, between about 4.5 ppm and about 5.0 ppm, between about5.0 ppm and about 6.0 ppm, between about 6.0 ppm and about 7.0 ppm,between about 7.0 ppm and about 8.0 ppm, between about 8.0 ppm and about10 ppm, between about 10 ppm and about 15 ppm, between about 15 ppm andabout 20 ppm, between about 20 ppm and about 30 ppm, or between about 30ppm and about 50 ppm (and overlapping ranges thereof) to reduce theproduction of non-PHA materials relative to the production of PHAmaterials in a culture. In another embodiment, the concentration ofcarbon dioxide or dissolved carbon dioxide is manipulated to betweenabout 0.01 ppm and about 0.05 ppm, between about 0.05 ppm and about 0.1ppm, between about 0.1 ppm and about 0.5 ppm, between about 0.5 ppm andabout 1.0 ppm, between about 1.0 ppm and about 1.5 ppm, between about1.5 ppm and about 1.75 ppm, between about 1.75 ppm and about 2.0 ppm,between about 2.0 ppm and about 2.5 ppm, between about 2.5 ppm and about3.0 ppm, between about 3.0 ppm and about 3.5 ppm, between about 3.5 ppmand about 4.0 ppm, between about 4.0 ppm and about 4.5 ppm, betweenabout 4.5 ppm and about 5.0 ppm, between about 5.0 ppm and about 6.0ppm, between about 6.0 ppm and about 7.0 ppm, between about 7.0 ppm andabout 8.0 ppm, between about 8.0 ppm and about 10 ppm, between about 10ppm and about 15 ppm, between about 15 ppm and about 20 ppm, betweenabout 20 ppm and about 30 ppm, or between about 30 ppm and about 50 ppmbetween about 50 ppm and about 100 ppm, between about 100 ppm and about200 ppm, between about 200 ppm and about 500 ppm, between about 500 ppmand about 1000 ppm, between about 100 ppm and about 1500 ppm, betweenabout 1500 ppm and about 2000 ppm, between about 2000 ppm and about 3000ppm, between about 3000 ppm and about 5000 ppm, between about 5000 ppmand about 10,000 ppm, between about 10,000 ppm and about 20,000 ppm (andoverlapping ranges thereof) to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inanother embodiment, the concentration of nitrogen or dissolved nitrogenis manipulated to above at least between about 0.01 ppm and about 0.05ppm, between about 0.05 ppm and about 0.1 ppm, between about 0.1 ppm andabout 0.5 ppm, between about 0.5 ppm and about 1.0 ppm, between about1.0 ppm and about 1.5 ppm, between about 1.5 ppm and about 1.75 ppm,between about 1.75 ppm and about 2.0 ppm, between about 2.0 ppm andabout 2.5 ppm, between about 2.5 ppm and about 3.0 ppm, between about3.0 ppm and about 3.5 ppm, between about 3.5 ppm and about 4.0 ppm,between about 4.0 ppm and about 4.5 ppm, between about 4.5 ppm and about5.0 ppm, between about 5.0 ppm and about 6.0 ppm, between about 6.0 ppmand about 7.0 ppm, between about 7.0 ppm and about 8.0 ppm, betweenabout 8.0 ppm and about 10 ppm, between about 10 ppm and about 15 ppm,between about 15 ppm and about 20 ppm, between about 20 ppm and about 30ppm, or between about 30 ppm and about 50 ppm, and overlapping rangesthereof, to reduce the production of non-PHA materials relative to theproduction of PHA materials in a culture. Without being limited bytheory, it is believed that, in some metabolic pathways, an increase inthe concentration of methane, oxygen, carbon dioxide, and/or nitrogencauses a metabolic shift that favors the production of PHA at theexpense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars, and/orlipids, particularly, but not necessarily, under growth-limiting, thatis, PHA synthesis, conditions. In one embodiment, the synthesis ofpolyhydroxyalkanoate (PHA) in a biomass material is effected, comprisingthe steps of: (a) providing a medium comprising a biomass metabolizing asource of carbon, and (b) increasing or maintaining above a minimum theconcentration of an element in the medium to cause the biomass tosynthesize PHA or increase the synthesis rate of PHA relative to thesynthesis rate of non-PHA material. In one embodiment, the PHA ispolyhydroxybutyrate (PHB). In one embodiment, the biomass is one or moremicroorganisms. In one embodiment, the step of increasing theconcentration of the element in the medium causes a reduction in theconcentration of sugar, lipids, nucleic acids, saccharides,polysaccharides, and/or pigments in the biomass relative to theconcentration of PHA in the biomass. In one embodiment, the element isone or more of the following: phosphorus, phosphate, phosphoric acid,sodium phosphate, disodium phosphate, monosodium phosphate, or potassiumphosphate, methane, oxygen, carbon dioxide, hydroxyl ions, hydrogen ion,nitrogen. In one embodiment, the element is one or more of thefollowing: EDTA, citric acid, iron, copper, magnesium, manganese, zinc,calcium, potassium, boron. In one embodiment, the PHA synthesis rate isincreased relative to the synthesis rate of PHA in said biomass in theabsence of said. In one embodiment, the synthesis ofpolyhydroxyalkanoate (PHA) in a biomass material is effected, comprisingthe steps of: (a) providing a medium comprising a biomass and anelement, and (b) maintaining above a minimum concentration or increasingthe concentration of the element in the medium to cause the biomassmaterial to metabolically synthesize PHA at the expense of alternativebiomass energy and/or carbon storage materials. In one embodiment, theelement is phosphorus, oxygen, magnesium, calcium, copper, iron,methane, carbon dioxide, or nitrogen. In one embodiment, the element isphosphorus. In one embodiment, the element is oxygen. In one embodiment,the element is magnesium. In one embodiment, the element is calcium. Inone embodiment, the element is copper. In one embodiment, the element isiron. In one embodiment, the biomass comprises one or moremicroorganisms. In one embodiment, one or more microorganisms comprisemethanotrophic microorganisms. In one embodiment, one or moremicroorganisms comprise heterotrophic microorganisms. In one embodiment,one or more microorganisms comprise autotrophic microorganisms. In oneembodiment, one or more microorganisms comprise methanogenicmicroorganisms.

In one embodiment, the invention comprises manipulating theconcentration of elements, e.g., copper, iron, phosphorus, oxygen,methane, carbon dioxide, in the culture medium to control theconcentration of sMMO and/or pMMO produced by a methanotrophic culturein order to control the relative ratio of sMMO to pMMO in the cultureand thereby control the growth conditions, metabolic status, metabolicdisposition, and/or specification of PHA produced by the culture. Insome embodiments, sMMO is expressed in a range between about 0% and 100%of a methanotrophic culture by dry cell weight, as a percentage ofmicroorganisms expressing sMMO, or as a percentage of total MMOexpressed by one or more methanotrophic cells, including between 0% and1%, between about 1% and about 2%, between about 2% and about 3%,between about 3% and about 5%, between about 5% and about 10%, betweenabout 10% and about 20%, between about 20% and about 30%, between about30% and about 50%, between about 50% and about 70%, between about 70%and about 80%, between about 80% and about 90%, between about 90% andabout 95%, between about 95% and about or 100%, and overlapping rangesthereof. Simultaneously, or independently, in some embodiments, pMMO isexpressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingpMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout or 100%, and overlapping ranges thereof. In some embodiments, theratio of sMMO to pMMO produced in a methanotrophic culture is controlledto control the specification of PHA produced by a culture In someembodiments, the relative weight ratio of sMMO to pMMO in amethanotrophic culture is at least or approximately 0 to 1,approximately 0.0000001 to 1, approximately 0.0001 to 1, approximately0.001 to 1, approximately 0.01 to 1, approximately 0.1 to 1,approximately 1 to 1, approximately 2 to 1, approximately 3 to 1,approximately 5 to 1, approximately 10 to 1, approximately 15 to 1,approximately 20 to 1, approximately 25 to 1, approximately 30 to 1,approximately 35 to 1, approximately 50 to 1, approximately 65 to 1,approximately 70 to 1, approximately 80 to 1, approximately 90 to 1,approximately 95 to 1, approximately 98 to 1, approximately 99 to 1,approximately 100 to 1, approximately 1000 to 1, approximately 10,000 to1, approximately 100,000 to 1, or approximately 1,000,000 to 1,respectively. In some embodiments, the relative weight ratio of pMMO tosMMO in a methanotrophic culture is approximately 0 to 1, approximately0.0000001 to 1, approximately 0.0001 to 1, approximately 0.001 to 1,approximately 0.01 to 1, approximately 0.1 to 1, approximately 1 to 1,approximately 2 to 1, approximately 3 to 1, approximately 5 to 1,approximately 10 to 1, approximately 15 to 1, approximately 20 to 1,approximately 25 to 1, approximately 30 to 1, approximately 35 to 1,approximately 50 to 1, approximately 65 to 1, approximately 70 to 1,approximately 80 to 1, approximately 90 to 1, approximately 95 to 1,approximately 98 to 1, approximately 99 to 1, approximately 100 to 1,approximately 1000 to 1, approximately 10,000 to 1, approximately100,000 to 1, or approximately 1,000,000 to 1. In some embodiments,controlling the relative concentrations of sMMO and pMMO produced by aculture of methanotrophic microorganisms, it is possible to control themetabolic status of the culture and thereby control the type of PHA andother cellular material produced by the culture, particularly in thepresence of volatile organic compounds, fatty acids, volatile fattyacids, methanol, formate, acetate, dissolved carbon dioxide, dissolvedmethane, dissolved oxygen, and other elements or compounds that impactthe metabolism of a culture of methanotrophic microorganisms in acertain manner according to the relative concentration of sMMO or pMMOin such a culture. In some embodiments, sMMO and/or pMMO is expressed ina range between about 0% and 100% of a methanotrophic culture by drycell weight, as a percentage of microorganisms expressing sMMO or pMMO,or as a percentage of total MMO expressed by one or more methanotrophiccells, including between 0% and 1%, between about 1% and about 2%,between about 2% and about 3%, between about 3% and about 5%, betweenabout 5% and about 10%, between about 10% and about 20%, between about20% and about 30%, between about 30% and about 50%, between about 50%and about 70%, between about 70% and about 80%, between about 80% andabout 90%, between about 90% and about 95%, between about 95% and aboutor 100%, and overlapping ranges thereof prior to, during, throughout, orafter a PHA production phase. In one embodiment, sMMO is not expressed,or is expressed in low concentrations, in a methanotrophic culture priorto, during, throughout, or after a PHA production phase. Without beinglimited by theory, it is believed that the directed or controlledabsence or reduction of sMMO in a methanotrophic culture producing PHA,particularly in the presence of non-methane organic compounds that canbe metabolized by methanotrophic microorganisms, engenders PHAproduction stability, consistency, and control by selectively shieldingagainst the metabolism of one or some or many non-methane compounds thatmight otherwise be metabolized in the presence of sMMO, which enablesthe metabolism of a larger group of non-methane compounds than pMMO.Similarly, in one embodiment, pMMO is not expressed, or is expressed inlow concentrations, in a methanotrophic culture prior to, during,throughout, or after a PHA production phase. In some embodiments, thedirected or controlled absence or reduction of pMMO in a methanotrophicculture producing PHA, particularly in the presence of non-methaneorganic compounds that can be metabolized by methanotrophicmicroorganisms, engenders PHA production stability, consistency, andcontrol by selectively inducing or promoting the metabolism of one orsome or many non-methane compounds that might otherwise be not bemetabolized using pMMO. In some embodiments, in some methanotrophiccultures, sMMO promotes PHA synthesis at high intracellularconcentrations by reducing cellular production of non-PHA materials,particularly as compared to PHA synthesis using pMMO. By controlling theconcentration of sMMO relative to pMMO in a methanotrophic microorganismculture in the presence of methane and/or non-methane organic compounds,including VOCs and other carbon-containing materials, such as volatilefatty acids, acetone, acetic acid, acetate, formate, formic acid,chloroform, methylene chloride, carbon dioxide, ethane, and/or propane,it is possible to control the specification or type of PHA produced bythe culture, including the molecular weight, polydispersity, melt flow,impact strength, and other similar functional characteristics. In someembodiments, it is preferable to maintain the concentration of copper inthe culture media in order to promote sMMO production. In someembodiments, in some methanotrophic cultures, the production of sMMO inmany, most, or substantially all of the methanotrophic cells enables theculture to produce more PHA when subject to a nutrient limiting stepthan would otherwise be produced if the relative ratio of pMMO in theculture was higher prior to the nutrient limiting step. Conversely, insome embodiments, it is preferable to maintain the concentration ofcopper in the culture media in order to promote pMMO production. In someembodiments, in some methanotrophic cultures, the production of pMMO inmany, most, or substantially (e.g., more than 50%, more than 60%, morethan 70%, more than 80%, more than 90%, or more) all of themethanotrophic cells enables the culture to produce more PHA whensubject to a nutrient limiting step than would otherwise be produced ifthe relative ratio of sMMO in the culture was higher prior to thenutrient limiting step. In one embodiment, one or more methanotrophiccells or cultures are subject to repeated growth and PHA synthesiscycles or steps, wherein the relative concentration of sMMO to pMMO inthe cells or cultures are caused to remain approximately similar or thesame in each new cycle or step in order to control the specificationand/or functionality of the PHA produced by the culture. In severalembodiments, the relative concentration of sMMO to pMMO is variableacross the repetitions. In some embodiments, the ratio of relativeconcentrations decreases (e.g., there is progressively less sMMO andprogressively more pMMO with each repetition). In one embodiment, thefunctional characteristics of PHA produced by a methanotrophic cultureexposed to methane emissions comprising methane, carbon dioxide, and oneor more volatile organic compounds are controlled and optimized, whereinthe method comprises: (a) providing a methanotrophic culture in amineral medium comprising nutrients, (b) controlling the concentrationof one or more of nutrients in the medium to cause the culture toproduce a defined relative ratio of sMMO to pMMO, (c) and controllingthe concentration of the nutrients in the medium to cause the culture toproduce PHA. In one embodiment, the culture produces essentially onlypMMO. As used herein, the term “essentially only” shall be given itsordinary meaning and shall also refer to production of pMMO or sMMO inan amount greater than about 50%, greater than about 55%, greater thanabout 60%, greater than about 65%, greater than about 70%, greater thanabout 75%, greater than about 80%, greater than about 85%, greater thanabout 90%, greater than about 95%, greater than about 97%, or greaterthan about 99% of the monooxygenase produced by a culture. In otherembodiments, essentially only shall refer to the ratio of production ofpMMO to sMMO. In some embodiments wherein a culture is producingessentially only pMMO, the ration of pMMO to sMMO is about 2:1, about5:1, about 10:1 about 50:1, about 100:1, about 250:1, about 500:1, about1000:1, about 2500:1, about 5000:1, about 10,000:1, or greater. In stilladditional embodiments, the culture may produce both sMMO and pMMO, butwith respect to the activity of the enzymes, the culture produces PHAessentially only through the pMMO-mediated pathway. In one embodiment,therefore, even if the culture comprises an equal concentration of sMMOand pMMO, PHA may be preferentially produced via pMMO. In otherembodiments, depending on the culture conditions and/or themicroorganism strain, the inverse can occur (e.g., the culture producesessentially only sMMO). In one embodiment, the concentration of sMMO inthe culture is more than 2 times greater than the concentration of pMMOin the culture. In one embodiment, the concentration of sMMO is morethan 5 times greater than the concentration of pMMO in the culture. Inone embodiment, the concentration of sMMO is more than 10 times greaterthan the concentration of pMMO in culture. In one embodiment, theconcentration of pMMO is more than 2 times greater than theconcentration of sMMO in said culture. In one embodiment, theconcentration of pMMO is more than 5 times greater than theconcentration of sMMO in said culture. In one embodiment, steps (a)through (c) are repeated, wherein the concentration of sMMO relative topMMO in the culture is substantially the same (e.g., within 5%, 10%,25%, 50% or 75% relative proportion by weight) in step (c) in at leasttwo repetitions.

In one embodiment, a method is provided for converting acarbon-containing gas or material into a polyhydroxyalkanoate (PHA) athigh efficiency, comprising: (a) providing a methanotrophic culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture wherein the gene encoding the soluble methane monooxygenaseenzyme is absent in the one or more microorganisms, (d) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d). Copperis a critical component of many methanotrophic systems, including bothsMMO and pMMO, and copper typically controls the switch between sMMO andpMMO production. Reducing copper concentration (below 0.1, 1, 2, 4, 10,20, 40, 100 micromolar, below 0.001, 0.01, 0.1, 1, 2, 4, 8, 10, 15, 20,40, 100, 200 mg/L, or below 0.001, 0.01, 0.1, 1, 2, 4, 10, 100 mg/g dryweight of microorganism biomass) typically increases sMMO production,while increasing copper concentration (above 0.1, 1, 2, 4, 10, 20, 40,100 micromolar, above 0.001, 0.01, 0.1, 1, 2, 4, 8, 10, 15, 20, 40, 100,200 mg/L, or, or above 0.001, 0.01, 0.1, 1, 2, 4, 10, 100 mg/g dryweight of microorganism biomass) typically increases pMMO production.Copper is generally added to a culture each time water or mineral mediais added to a culture, since trace copper is difficult to remove fromeven purified water, and copper is needed for methanotrophic cellularreplication/growth, since MMO generally drives the oxidation of methaneto biomass, and MMO is a copper-containing enzyme. Applicant hassurprisingly discovered that microorganisms that do not possess orexpress the gene for sMMO unexpectedly produce PHA at high efficiency,and can be selectively cultured, using culture selection pressures, toout-compete microorganisms that do possess or express the gene for sMMO(particularly in non-sterile systems, wherein new microorganisms areperiodically introduced to the culture) by limiting, controlling, orreducing copper concentrations and simultaneously subjecting the cultureto growth-polymerization-growth repetitions, as described herein. In oneembodiment, microorganisms that possess higher concentrations of PHAswitch from polymerization to growth mode (wherein the microorganismsproduce soluble and/or particulate methane monooxygenase) more quicklyand efficiently than microorganisms that possess lower concentrations ofPHA, and/or carry out cellular replication more efficiently in general;by reducing copper concentrations to levels that would traditionallycause the culture to express sMMO (e.g., less than 0.001, 0.01, 0.1, 1,10, or 100 mg/L, or less than 0.01, 0.1, 1, 10, or 100 micromolar, orless than 0.001, 0.01, 0.1, 1, 10, or 100 mg per gram microorganism dryweight) while also cycling between growth and polymerization cycles,microorganisms that produce high concentrations of PHA and also growquickly in low copper concentrations out-compete microorganisms thatproduce less PHA and grow slower in low copper concentrations. SincepMMO renders a faster metabolism than sMMO, by reducing the copperconcentration in the medium (permanently or temporarily) totraditionally sMMO-generating concentrations, and concurrentlysubjecting a culture to growth-polymerization-growth repetitions whichselect for microorganisms that generate high PHA concentrations andmetabolize efficiently in transitioning from polymerization mode togrowth mode, Applicant has discovered that a high-efficiencymicroorganism can be selectively produced and maintained, including innon-sterile conditions, that does not contain or express the geneticcoding for sMMO, accumulates high concentrations of PHA, andout-competes microorganisms that produce sMMO. The ability to cause amethanotrophic culture to generate microorganisms that do not possess orexpress the gene encoding sMMO by reducing copper concentrations is asurprising and unexpected result, and offers a range of advantages,including superior process stability (microorganisms produce only pMMO,regardless of copper concentration), PHA consistency (the metabolicpathway, pMMO, is unchanging, thereby controlling the characteristics ofPHA produced, such as monomer composition, molecular weight,polydispersity, elongation, modulus, viscosity), and increased metabolicefficiency (e.g., rate, oxidation, metabolism, copper requirements). Inaddition to copper, other nutrients can be used, either individually orin combination, in similar fashion to control for the selectiveproduction of microorganisms that do not possess or express the geneencoding soluble methane monooxygenase, such nutrients including:methane, oxygen, phosphorus, magnesium, iron, boron, aluminum, calcium,cobalt, chloride, chromium, EDTA, manganese, molybdenum, sulfur, nickel,zinc, and/or potassium. In one embodiment, more than 10%, 25%, 50%, 75%,or 80% of the culture does not contain or express the gene encodingsoluble methane monooxygenase.

In one embodiment, a method is provided for converting acarbon-containing material (e.g., methane, carbon dioxide, propane,ethane, acetone, acetate, formaldehyde, a volatile organic compound, anon-methane organic compound, carbon dioxide) into apolyhydroxyalkanoate (PHA), the method comprising: (a) providing amethanotrophic culture, (b) providing a medium comprising one or morenutrient comprising a carbon-containing material that can be metabolizedby the culture, (c) controlling the concentration of the one or morenutrient in the medium to cause the cellular replication of the culturew wherein the gene encoding the ethylmalonyl-CoA pathway is present orexpressed in one or more microorganism in said culture, (d) controllingthe concentration of said one or more nutrients in said medium to causesaid culture to produce PHA, and (e) repeating steps (a) through (d).Applicant has surprisingly discovered that the ethylmalonyl-CoA (EMC)pathway, combined with required and controlled conditions, enables theproduction of over 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, and 95%intracellular PHA concentrations. Applicant has also surprisinglydiscovered that the ethylmalonyl-CoA (EMC) pathway can enablemethanotrophic microorganisms to switch from growth to PHApolymerization and back to growth at high efficiency (that is, at higherefficiency than microorganisms that do not express or utilize the EMCpathway) even when intracellular PHA concentrations exceed over 50%,55%, 60%, 65%, 70%, 80%, 85%, 90%, and 95% by weight, whereas manycultures with PHA concentrations exceeding 50%, 60%, or 70% cannoteffectively return to growth mode following a polymerization period. Inseveral embodiments, the increased growth efficiency of microorganismsexpressing the EMC pathway (as compared to those microorganisms that donot express the pathway) is greater than about 1.1-fold, 1.2-fold,1.5-fold, 2-fold, about 4-fold, about 6-fold, about 8-fold, about10-fold, or greater. Thus, by subjecting a culture togrowth-polymerization-growth cycling, Applicant has found that it ispossible to select for preferential growth of microorganisms thatpossess the gene encoding the EMC pathway. In addition, Applicant hassurprisingly discovered that it is possible to selectively produce aculture of microorganisms that express the EMC pathway in sterile ornon-sterile conditions, including in the presence of methane,non-methane carbon-containing materials, or other materials thatinfluence the metabolism of the microorganisms, by simultaneouslysubjecting the culture to growth-PHA polymerization-growth cycling, asdescribed herein, while also controlling the concentration one or morenutrients available to the culture. In one embodiment, the concentrationof a nutrient, such as copper, is controlled to selectively favor theproduction of EMC-pathway microorganisms. In some embodiments,microorganisms that do not possess or express the genetic materialencoding soluble methane monooxygenase also possess the genes encodingthe EMC-pathway. As used herein the terms “genetic material” and“nucleic acid” shall be given their ordinary meanings and shall alsorefer to polymer of nucleotides. Non-limiting examples thereof includeDNA (e.g. genomic DNA, cDNA), RNA molecules (e.g. mRNA) and chimerasthereof. The nucleic acid molecule can be obtained by cloning techniquesor synthesized. DNA can be double-stranded or single-stranded (codingstrand or non-coding strand (e.g., antisense)). Also, as used herein,the term “expression” shall be given its ordinary meaning and shall beunderstood to define the process by which a gene is transcribed intomRNA (transcription) and the mRNA is then be translated (translation)into one polypeptide (or protein) or more.

Thus, as described herein, by subjecting the culture togrowth-polymerization-growth (GPG) cycling while also controlling orreducing the concentration of copper, or other nutrients that impact theswitch between sMMO and pMMO production, it is possible to selectivelyproduce microorganisms that do not possess or express the geneticmaterial encoding soluble methane monooxygenase but do possess the geneencoding the EMC pathway. The ability to selectively producemicroorganisms that contain or express the genes encoding the EMCpathway and/or do not contain or express the genes encoding sMMO offerssignificant process advantages, including process stability(microorganisms produce consistent concentrations of PHA and produceonly pMMO, regardless of copper concentration or microorganismcontamination), PHA consistency (the metabolic pathway is unchanging,thereby controlling the characteristics of PHA produced, such as monomercomposition, molecular weight, polydispersity, elongation, modulus,viscosity), and increased metabolic efficiency (e.g., rate, oxidation,metabolism, nutrient requirements). In some embodiments, suchmicroorganisms and processes may be combined with other microorganismsand processes, wherein a culture may contain microorganisms containinggenes encoding or expressing both sMMO and pMMO and genetic packagesthat do not encode or express the EMC pathway; in some embodiments,microorganisms that do not possess or express one or more gene encodingsMMO comprise less than 1%, less than 5%, less than 10%, less than 20%,less than 50%, less than 75%, more than 75% of a culture; in otherembodiments, microorganisms that contain or express genetic materialencoding the EMC pathway comprise less than 1%, less than 5%, less than10%, less than 20%, less than 50%, less than 75%, more than 75% of aculture.

In one embodiment, a method is provided for converting acarbon-containing gas or material into a polyhydroxyalkanoate (PHA) athigh efficiency, comprising: (a) providing a methanotrophic culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture wherein the gene encoding the soluble methane monooxygenaseenzyme is absent in the one or more microorganisms, (d) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d). Copperis a critical component of many methanotrophic systems, including bothsMMO and pMMO, and copper typically controls the switch between sMMO andpMMO production. In several embodiments, reducing copper concentration(below about 0.1, about 1, about 2, about 4, about 10, about 20, about40, about 100 micromolar, below about 0.0001, about 0.001, about 0.01,about 1, about 2, about 4, about 8, about 10, about 15, about 20, about40, about 100, about 200 mg/L, or below about 0.001, about 0.01, about0.1, about 1, about 2, about 4, about 10, about 100 mg/g dry weight ofmicroorganism biomass) typically (or for at least some methanotrophicmicroorganisms) increases sMMO production. In contrast, increasingcopper concentration (above about 0.1, about 1, about 2, about 4, about10, about 20, about 40, about 100 micromolar, above about 0.001, about0.01, about 0.1, about 1, about 2, about 4, about 8, about 10, about 15,about 20, about 40, about 100, about 200 mg/L, or, or above about 0.001,about 0.01, about 0.1, about 1, about 2, about 4, about 10, about 100mg/g dry weight of microorganism biomass) typically (or for at leastsome methanotrophic microorganisms) increases pMMO production. Copper isgenerally added to a culture each time water or mineral media is addedto a culture, since trace copper is difficult to remove from evenpurified water, and copper is needed for methanotrophic cellularreplication/growth, since MMO generally drives the oxidation of methaneto biomass, and MMO is a copper-containing enzyme.

In accordance with several embodiments, Applicant has surprisinglydiscovered that selective culture conditions can be employed that allowfor the dominance of a culture by microorganisms that utilize the pMMOpathway, even at low or reduced copper concentrations (e.g., thoseconditions in which sMMO would typically be produced). In someembodiments, the pMMO pathway or enzyme is the exclusive pathway orenzyme expressed or used in the microorganism for the production ofmethane monooxygenase (e.g., the selective pressures of the processesdisclosed herein induce a loss of the genetic material encoding the sMMOgene in the culture or microorganism over time, or the selectivepressures result in the dominance or growth or metabolic success ofmicroorganisms that do not possess or express the genetic material usedfor sMMO production). In some embodiments, the pMMO pathway is eitherpreferentially expressed or preferentially active in the culture, suchthat the microorganisms still retain the genetic material necessary toproduce sMMO, but sMMO is either not produced, produced but not used,produced and functionally blocked (e.g., negative feedback mechanisms),produced and functionally deficient under the culture conditions, and/orproduced but metabolically outcompeted for substrate by pMMO enzymes.Thus, in several embodiments, microorganisms that have reduced sMMOexpression or function unexpectedly produce PHA at high efficiency, andcan be selectively cultured, using culture selection pressures, toout-compete microorganisms that do express sMMO (particularly innon-sterile systems, wherein new microorganisms are periodicallyintroduced to the culture) by limiting, controlling, or reducing copperconcentrations and simultaneously subjecting the culture togrowth-polymerization-growth repetitions, as described herein.

In one embodiment, microorganisms that possess higher concentrations ofPHA switch from polymerization to growth mode (wherein themicroorganisms produce soluble and/or particulate methane monooxygenase)more quickly and efficiently than microorganisms that possess lowerconcentrations of PHA, and/or carry out cellular replication moreefficiently in general. By reducing copper concentrations to levels thatwould cause or enable, or traditionally cause or enable, the culture, orat least one or more methanotrophic microorganisms, to express sMMO(e.g., less than about 0.001, about 0.01, about 0.1, about 1, about 10,or about 100 mg/L, or less than about 0.01, about 0.1, about 1, about10, or about 100 micromolar, or less than about 0.001, about 0.01, about0.1, about 1, about 10, or about 100 mg per gram microorganism dryweight) while also cycling between growth and polymerization cycles,microorganisms that produce high concentrations of PHA and also growquickly in low copper concentrations out-compete microorganisms thatproduce less PHA and grow slower in low copper concentrations. SincepMMO renders a faster metabolism than sMMO, by reducing the copperconcentration in the medium (permanently or temporarily) as compared totraditionally pMMO-generating copper concentrations (e.g., toconcentrations that could enable or induce one or more methanotrophicmicroorganisms to produce sMMO if present in the medium), andconcurrently (or subsequently) subjecting a culture togrowth-polymerization-growth repetitions which select for microorganismsthat generate high PHA concentrations and metabolize efficiently intransitioning from polymerization mode to growth mode, Applicant hasdiscovered that a high-efficiency microorganism can be selectivelyproduced and maintained (even in non-sterile conditions) that eitherdoes not contain, does not express, does not produce, or expresses orproduces at reduced levels sMMO, accumulates high concentrations of PHA,and out-competes microorganisms that produce sMMO. The ability to causea methanotrophic culture to generate microorganisms that do not possessthe genetic material encoding sMMO or express sMMO at reduced levels (orreduced functionality) by reducing copper concentrations is a surprisingand unexpected result, and offers a range of advantages. For example,superior process stability (microorganisms produce only pMMO, regardlessof copper concentration), PHA consistency (the metabolic pathway, pMMO,is unchanging, thereby controlling the characteristics of PHA produced,such as monomer composition, molecular weight, polydispersity,elongation, modulus, viscosity), and increased metabolic efficiency(e.g., rate, oxidation, metabolism, copper requirements) are achieved.In addition to copper, other nutrients can be used, either individuallyor in combination, in similar fashion to control for the selectiveproduction of microorganisms that do not possess or express the geneencoding soluble methane monooxygenase, such nutrients including:methane, oxygen, phosphorus, magnesium, iron, boron, aluminum, calcium,cobalt, chloride, chromium, EDTA, manganese, molybdenum, sulfur, nickel,zinc, and/or potassium. In one embodiment, more than 10%, 25%, 50%, 75%,or 80% of the culture does not contain the gene encoding soluble methanemonooxygenase.

In one embodiment, a method is provided for converting acarbon-containing material (e.g., methane, carbon dioxide, propane,ethane, acetone, acetate, formaldehyde, a volatile organic compound, anon-methane organic compound, carbon dioxide) into apolyhydroxyalkanoate (PHA), the method comprising: (a) providing amethanotrophic culture, (b) providing a medium comprising one or morenutrient comprising a carbon-containing material that can be metabolizedby the culture, (c) controlling the concentration of the one or morenutrient in the medium to cause the cellular replication of the culturewherein the genetic material encoding the ethylmalonyl-CoA (EMC) pathway(e.g., the various enzymes or co-factors that are involved in thepathway) is present in one or more microorganism in said culture, (d)controlling the concentration of said one or more nutrients in saidmedium to cause said culture to produce PHA, and (e) repeating steps (a)through (d). Applicant has surprisingly discovered that the EMC pathway,combined with required and controlled conditions, enables the productionof over 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, and 95% intracellularPHA concentrations. Applicant has also surprisingly discovered that themicroorganisms using the EMC pathway can be switched from growthconditions or metabolism to PHA polymerization conditions or metabolismand back to growth conditions or metabolism at high efficiency, evenwhen intracellular PHA concentrations exceed over 50%, 55%, 60%, 65%,70%, 80%, 85%, 90%, and 95% by weight, whereas many cultures with PHAconcentrations exceeding 50%, 60%, or 70% cannot effectively return togrowth mode following a polymerization period. Thus, by subjecting aculture to growth-polymerization-growth cycling, Applicant has foundthat it is possible to select for microorganisms that possess the geneencoding the EMC pathway. In addition, Applicant has surprisinglydiscovered that it is possible to selectively produce a culture ofmicroorganisms that express the EMC pathway in sterile or non-sterileconditions, including in the presence of methane, non-methanecarbon-containing materials, or other materials that influence themetabolism of the microorganisms, by simultaneously subjecting theculture to growth-PHA polymerization-growth cycling, as describedherein, while also controlling the concentration one or more nutrientsavailable to the culture. In one embodiment, the concentration of anutrient, such as copper, is controlled to selectively favor theproduction of EMC-pathway microorganisms. In some embodiments,microorganisms that do not possess the genetic material encoding solublemethane monooxygenase (or have the genetic material but express atreduced levels or at reduced levels of activity) also possess the genesencoding the EMC-pathway. Thus, as described herein, by subjecting theculture to growth-polymerization-growth (GPG) cycling while alsocontrolling or reducing the concentration of copper, or other nutrientsthat impact the switch between sMMO and pMMO production, it is possibleto selectively produce microorganisms that do not possess the geneencoding soluble methane monooxygenase (or have the genetic material butexpress sMMO at reduced levels or at reduced levels of activity) but dopossess the gene encoding the EMC pathway. The ability to selectivelyproduce microorganisms that contain the genetic material for and expressthe gene encoding the EMC pathway and/or do not contain the geneticmaterial for or do not express the gene encoding sMMO offers significantprocess advantages, including process stability (microorganisms produceconsistent concentrations of PHA and produce only pMMO, regardless ofcopper concentration or microorganism contamination), PHA consistency(the metabolic pathway, is unchanging, thereby controlling thecharacteristics of PHA produced, such as monomer composition, molecularweight, polydispersity, elongation, modulus, viscosity), and increasedmetabolic efficiency (e.g., rate, oxidation, metabolism, nutrientrequirements). In some embodiments, such microorganisms and processesmay be combined with other microorganisms and processes, wherein aculture may contain microorganisms containing genes encoding both sMMOand pMMO and genetic material that does not include material encodingthe EMC pathway; in some embodiments, microorganisms that do not possessgene(s) encoding sMMO comprise less than about 1%, less than about 5%,less than about 10%, less than about 20%, less than about 50%, less thanabout 75%. In other embodiments, microorganisms that contain geneticmaterial encoding the EMC pathway comprise less than about 1%, less thanabout 5%, less than about 10%, less than about 20%, less than about 50%,or less than about 75%. In some embodiments, microorganisms andprocesses may be combined with other microorganisms and processes,wherein a culture may contain microorganisms containing genes encodingboth sMMO and pMMO and genetic material that does not include materialencoding the EMC pathway. In several embodiments, additional selectionmethods, and/or “spiking” of a culture with microorganisms of a certaintype or genetic makeup can be used to achieve microorganism cultureswith desired characteristics/demographics.

Several embodiments of the invention comprise a culture or bacterium, oran isolated culture or bacterium, that (a) does not express or containthe genetic material encoding soluble methane monooxygenase, (b)expresses or contains the genetic material encoding the ethylmalonyl-CoApathway, and (c) produces polyhydroxyalkanoate (PHA) at intracellularconcentrations wherein the ratio of PHA to non-PHA biomass exceeds 3:1on a dry weight basis (e.g., wherein the concentration of PHA exceeds75% on a total dry cell weight basis). In some embodiments, such cultureor bacterium may be mixed with other cultures or bacteria, wherein suchculture or bacterium comprise, consists essentially of, or substantiallyexhibits the properties (a), (b), and (c). In several embodiments, theprocesses and methods disclosed herein are utilized to produce suchcultures and/or bacterium, including as part of a process to producePHA, including as a measure to continually drive selection pressures toproduce such cultures and/or bacterium, particularly, in someembodiments, in the presence of non-sterile input streams. In someembodiments, such culture(s) or bacterium is selected from a groupconsisting of: Methylosinus, Methylocystis, Methylococcus,Methylobacterium, and Pseudomonas. In some embodiments, the inventioncomprises the PHA produced by such a culture or bacterium, and/or thePHA comprising such a culture or bacterium.

Some embodiments of the invention comprise a methanotrophic culture orbacterium having (a) particulate methane monooxygenase activity in thepresence of copper ion concentrations between concentrations that cangenerate soluble methane monooxygenase in some methanotrophs, including0.001 micromolar and 1000 micromolar, (b) ethylmalonyl-CoA pathwayexpression in the presence of copper concentrations between 0.001micromolar and 1000 micromolar, and (c) intracellular concentrations ofpolyhydroxyalkanoate (PHA) wherein the ratio of PHA to non-PHA biomassexceeds 2.9:1 on a dry weight basis (e.g., greater than 25%, greaterthan 55%, greater than 59%, greater than 71%, greater than 72.5%,greater than 80%, greater than 85%, greater than 87%, greater than 90%,greater than 95%, greater than 98% as function of total dry cellweight). In some embodiment, such culture(s) or bacterium are selectedfrom a group consisting of: Methylosinus, Methylocystis, Methylococcus,Methylobacterium, and Pseudomonas. In some embodiments, such culture ofbacterium is a Type I, Type II, or Type X methanotroph. In someembodiments, such culture of bacterium is a Type I methanotroph. In someembodiments, such culture of bacterium is a Type II methanotroph. Insome embodiments, such culture of bacterium is a Type X methanotroph. Insome embodiments, such culture of bacterium is from a Methylosinus,Methylocystis, or Methylococcus genus. In some embodiments, such cultureof bacterium is from a Methylosinus genus. In some embodiments, suchculture of bacterium is from a Methylocystis genus. In some embodiments,such culture of bacterium is from a Methylococcus genus. In severalembodiments, the processes and methods disclosed herein are utilized toproduce such cultures and/or bacterium, including as part of a processto produce PHA, including as a measure to continually drive selectionpressures to produce such cultures and/or bacterium, particularly, insome embodiments, in the presence of non-sterile input streams. In someembodiments, such culture or bacterium is a mutant,genetically-engineered mutant, genetically-manipulated variant, and/orselection-pressure-induced mutant selected from a group consisting of:Methylosinus, Methylocystis, Methylococcus, Methylobacterium, andPseudomonas. In some embodiments, such culture of bacterium is a mutantof a strain in the Methylosinus genus. In some embodiments, such cultureof bacterium is a mutant of a strain in the Methylocystis genus. In someembodiments, such culture of bacterium is a mutant of a strain in theMethylococcus genus. In some embodiments, such culture of bacterium is amutant of a strain in the Methylobacterium genus. In some embodiments,such culture of bacterium is a mutant of a strain in the Pseudomonasgenus. In some embodiments, the invention comprises the PHA produced bysuch a culture or bacterium, and/or the PHA comprising such a culture orbacterium.

In some embodiments, the invention comprises a method for producing ormutating a methanotrophic culture or bacterium that can producepolyhydroxyalkanoate (PHA) at intracellular concentrations exceeding 71%by weight in a non-sterile environment, the method comprising: (a)providing a culture broth comprising methane, a medium comprising one ormore nutrients comprising copper, and a methanotrophic culture orbacterium; (b) controlling the concentration of copper in the medium toa concentration that can enable methanotrophic microorganisms to producesMMO (e.g. less than 0.001, 0.01, 0.1, 1, 2, 5, 10, or 50 micromolar, orless than 0.001, 0.01, 0.1, 1, 2, 5, 10, 50, or 100 mg/cell dry weight,depending on the culture strain), (c) reducing the concentration of oneor more nutrient in said medium (e.g., nitrogen, oxygen, magnesium,phosphate, sulfate) to cause said culture or bacterium to produce PHA,(d) increasing the concentration of said one or more nutrient of step(c) to cause said culture or bacterium to reproduce using essentiallyonly pMMO, and (e) subjecting said culture or bacterium to a repetitionof steps (b), (c), and (d). In some embodiments, the invention comprisesthe PHA of the culture or bacterium of such a process, and/or the PHAcomprising the culture or bacterium of such a process.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 57% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 71% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 23% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 80% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 85% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 90% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In some embodiments, the invention comprises using microorganisms toproduce polyhydroxyalkanoate (PHA) comprising methanotrophicmicroorganisms having the following characteristics: (a) themicroorganisms produce no soluble methane monooxygenase, or noperceptible soluble methane monooxygenase, at any copper concentration(e.g., at non-detectable concentrations), or do not express or containthe genetic material encoding soluble methane monooxygenase; (b) themicroorganisms express the ethylmalonyl-CoA metabolic pathway; and (c)the microorganisms produce polyhydroxyalkanoate at intracellularconcentrations exceeding 95% by dry weight, wherein the microorganismsare selected from the a group consisting of Methylosinus, Methylocystis,Methylococcus, Methylobacterium, and Pseudomonas.

In several embodiments of the invention, methanol is added to a cultureof methanotrophic microorganisms utilizing a closed loop recycling gasstream comprising methane. In some embodiments, methanotrophicmicroorganisms are enabled to grow under conditions of, and consume,very low concentrations of methane by co-utilizing methanol as a carbonsubstrate. In the past, the growth of methanotrophic microorganisms wassignificantly reduced under low methane concentrations due to, amongother things, low mass transfer rates. In some embodiments, by theaddition of methanol in a closed loop gas recycling system, it ispossible to effect the substantially complete elimination of methane bymethanotrophic microorganisms.

In several embodiments of the invention, the diffusion of light isincreased in a liquid growth culture media by reducing the density ofthe liquid in a light path. In some embodiments the culture comprisesautotrophic microorganisms. In some embodiments, the application of gasbubbles into the media decreases the relative solids density of thelight path, thus enabling an increased diffusion of light into a liquidculture media from a given light intensity energy.

In several embodiments of the invention, a series of submerged lightrods are placed into a liquid culture to manipulate or adjust theculture conditions. In some embodiments, the culture comprisesautotrophic microorganisms. In some embodiments, the light rods functionto diffuse light, diffuse gas, act as static or dynamic mixers, assistin the circulation of a liquid culture media, and/or facilitate heatexchange through the circulation of a gas, liquid, and/or combinationthereof.

Traditionally, pH control in a microorganism growth system is difficultand/or costly to maintain. In some embodiments, pH is controlled byvarying the nitrogen source supplied to a microorganism growth systembetween pH-increasing and pH-reducing nitrogen sources, e.g., NO₃ ⁻ andNH₃ ⁺, respectively. In some embodiments, nitrogen sources are utilizedthat do not significantly affect the pH of the system, including, whenapplicable, complex nitrogen sources such as biomass. In additionalembodiments, a closed loop system is employed to reduce changes in pH.In some embodiments, respiration-generated carbon dioxidecounterbalances increases in pH caused by the utilization ofpH-increasing nitrogen sources, such as nitrates. In one embodiment,nitrogen fixation is used to add hydroxyl ions to the culture medium,which may or may not be counterbalanced by the addition of protons fromeither biological or chemical sources. In one embodiment, nitratefixation is used to add hydroxyl ions to the culture medium, which mayor may not be counterbalanced by the addition of protons from eitherbiological or chemical sources. In one embodiment, ammonia or ammoniumfixation is used to add protons to the culture medium, which may or maynot be counterbalanced by the addition of hydroxyl ions from eitherbiological or chemical sources.

A number of methods are known for the induction of gas into liquid,including static mixing, ejector mixing, propeller mixing, and/or acombination thereof. Simultaneously, it is also known that shear can behighly detrimental to microorganism growth, and can often impede orpermanently deactivate microorganism metabolism. Thus, mass transfer ina gas-based system is often limited by the need to counterbalancesufficient mixing with shear considerations. In several embodiments ofthe invention, a vessel comprising liquid culture media is mixed with agas, e.g. methane, under relatively high shear conditions, and thensubsequently transferred to a vessel comprising liquid culture mediamaintained under relatively low shear conditions. In some embodiments,microorganism growth is primarily induced in the low shear vessel. Insome embodiments, high gas transfer rates are effected in the first highshear vessel by mixing while performed in the low shear vessel bygaseous diffusion.

In another embodiment, a closed loop gas recycling system is maintained,wherein a vessel comprising gas-utilizing microorganisms is suppliedwith gas, wherein the gas is utilized by gas-utilizing microorganisms,and wherein the rate at which gas is added to the system is determinedby the rate at which the pressure in the vessel changes in accordancewith the conversion of gases into metabolic derivatives (such asbiomass, carbon dioxide, and water). For example, a vessel containingmethane-utilizing microorganisms may be pressurized to 60 psi with acombination of methane and oxygen; as the pressure in the system dropsin accordance with the metabolism of the methane-utilizingmicroorganisms, additional methane and oxygen is added to the systemsuch that the pressure of the vessel remains at 60 psi. In certainembodiments, higher or lower pressures are maintained. In someembodiments, the system is periodically flushed to remove carbondioxide. In some embodiments, autotrophic microorganisms and a lightinjection system may be added to the system in order to convert carbondioxide into additional oxygen, thereby substantially reducing oreliminating the need to flush the system and/or introduce oxygen. In oneembodiment, a device is provided that is capable of carrying outgas-based fermentation, methanotrophic metabolism, bioreaction,autotrophic metabolism, heterotrophic metabolism, and/orbiocatalyst-based metabolism at high efficiency, particularly using oneor more, and particularly at least two gases as nutrient (e.g., carbonand oxygen) input sources, measured in the following terms: 1) gascapture efficiency, 2) mass transfer efficiency (including in terms ofthe power required to transfer gas into aqueous/dissolved form), and 3)material synthesis (in terms of grams per liter per hour). In oneembodiment, a system is provided for gas input reactions (e.g., methaneand oxygen; oxygen and carbon dioxide; carbon dioxide and methane;methane, ammonia, and oxygen; methane, ammonia, oxygen, and dinitrogen;methane, carbon dioxide, and oxygen; or various combinations of suchinput gasses) that utilizes a system comprising multiple reactionvessels. In one embodiment, one or more vessel may be equipped with arotating mixer. In one embodiment, the rotating mixer may inducecavitation in the liquid medium. In one embodiment, such cavitation maycause acute induction of gas entrainment into the liquid medium,significantly increasing mass transfer induction. In one embodiment, gasmay be injected into one or more of the vessels behind the leading edgeof a moving material in liquid medium, in order to reduce and thenincrease the driving pressure of the gas injection. In one embodiment,the pressure of the liquid medium may be pulsed through periods of highpressure and low pressure to increase the mass transfer of gas intoliquid medium. In one embodiment, the pulsation of pressure in a liquidmedium may be employed, wherein the high pressure (e.g., up to 100 psi)period may have a duration from 0.001 seconds to 25 minutes, and whereinthe low pressure period (e.g., from −25 inches vacuum to 5 psi) may havea duration from 0.001 seconds to 25 minutes. In one embodiment, therapid induction of pressure pulsation may be effected by fitting avessel with a means of transferring acoustic energy into the vesselmedium. In one embodiment, the rapid induction of pressure pulsation maybe effected by fitting a vessel with a transducer. In one embodiment,the rapid induction of pressure pulsation may be effected by fitting avessel with one or more sonication means, wherein the liquid medium issonicated, wherein such sonication is diffused throughout a volumesufficient to avoid damage to microorganisms or enzymes in the liquidmedium. In one embodiment, silica gel or silica-based liquid is added tothe liquid medium to increase the solubility of methane and oxygen inthe liquid medium. In one embodiment, the reaction vessels comprisefully or partially enclosed vessels. In one embodiment, the reactionvessels comprise fully or partially-enclosed medium-containing volumesor medium-containing compartments, within or in addition to one or moretanks, compartments, vessels, or other volumes. In one embodiment, thevessels may be plastic or stainless steel enclosed vessels ormedium-containing volumes. In one embodiment, the vessels may not bephysically connected. In one embodiment, the vessels may be physicallyconnected. In one embodiment, gas may be directed into one or more ofthe vessels simultaneously. In one embodiment, a reactor, reactorsystem, or system may comprise multiple vessels combined. In oneembodiment, gas may be directed equally into each of vessels. In oneembodiment, gas may be directed more into one vessel and less intoanother vessel. In one embodiment, gas may be directed first into onevessel, and then into another vessel. In one embodiment, gas may beexhausted from all vessels equally. In one embodiment, gas may beexhausted from all vessels individually, or more from one vessel andless from another vessel. In one embodiment, exhaust gas may be directedfrom one vessel into another vessel. In one embodiment, the liquidmedium of the vessels is discrete and not mixed between the vessels. Inone embodiment, the liquid medium of the vessels is not discrete and ismixed between the vessels. In one embodiment, gas is directed equallyinto all vessels, and liquid medium is mixed between the vessels. In oneembodiment, gas is directed equally into all vessels, and liquid mediumis at least partially mixed between the vessels. In one embodiment, gasis directed equally into all vessels, and liquid medium is at leastpartially mixed between the vessels. In one embodiment, gas is directedequally into all vessels, and liquid medium is not mixed between thevessels. In one embodiment, gas is directed first into one vessel andthen into another vessel, and liquid medium is mixed between thevessels. In one embodiment, gas is directed first into one vessel andthen into another vessel, and liquid medium is not mixed between thevessels. In one embodiment, exhaust gas from a first vessel is directedinto a second vessel, and liquid medium is mixed between the vessels. Inone embodiment, gas is directed individually and discretely into eachvessel, and liquid medium is mixed between the vessels. In oneembodiment, exhaust gas is directed individually and discretely intoeach vessel, and liquid medium is not mixed between the vessels. In oneembodiment, the concentration of dissolved gas is caused to remainrelatively elevated in one vessel and relatively depressed in anothervessel. In one embodiment, the concentration of dissolved gas is causedto remain substantially equal in multiple vessels. In one embodiment,the gases are caused to be mixed equally throughout the vessels. In oneembodiment, the gases are caused to move sequentially through thevessels. In one embodiment, the gases are caused to be injectedindividually into through the vessels. In one embodiment, the gases arecaused to be injected individually and simultaneously into the vessels.In one embodiment, the gases are caused to move simultaneously throughthe vessels on an individual basis and medium is caused to not be fullymixed between the vessels, such that the medium remains substantiallyisolated. In one embodiment, the gases are caused to move sequentiallythrough the vessels and medium is caused to not be fully mixed betweenthe vessels. In one embodiment, the gases are caused to be injectedindividually and simultaneously into the vessels and medium is caused tobe mixed between the vessels, such that the medium remains substantiallynon-isolated. In one embodiment, the gases are caused to movesequentially through the vessels to cause the medium in the vessels tobe substantially non-isolated and gas is caused to move in multipledirections in each vessel. In one embodiment, the gases are caused to beinjected simultaneously into the vessels, such that the gases are causedto move the medium in the vessels to be substantially non-isolated. Inone embodiment, the gases are caused to be injected simultaneously intothe vessels in such a manner that the gases are not caused to move themedium between the vessels. In one embodiment, gas may be moved betweenvessels by mechanical means, such as a pump. In one embodiment, liquidmedium may be moved between vessels by mechanical means, such as a pump.In one embodiment, gas may be injected into a vessel by mechanicalmeans. In one embodiment, liquid medium may be injected into a vesselsby mechanical means, such as a pump. In one embodiment, gas and liquidmedium may be injected simultaneously into a vessel by mechanical means,such as a pump, nozzle, venturi, compressor, diffusor, vacuum. In oneembodiment, the vessels may be equipped with one or more internalcavitation mechanisms. In one embodiment, the vessels may be operatedunder recurring periods or patterns of pressure and vacuum to induceoptimal mass transfer efficiency. In one embodiment, the vessels may befilled with one or more materials, that are more or less dense thanliquid medium, that are able to dissolve or absorb high concentrationsor amounts of gases, such as methane, oxygen, or carbon dioxide, whereinsuch materials may be silica-based gels or beads, activated carbon,nickel-plated spheres, polypropylene beads, PES beads, PTFE beads, orultra high molecular weight polyethylene pellets. In one embodiment, therapid pulsation of pressure in the vessels causes the absorbentmaterials to absorb gases at high concentration, and then release atleast some of the gases into the medium, causing an increase in masstransfer into the liquid medium. In one embodiment, a vessel is filledwith liquid medium containing gas-absorbent material, such as plasticbeads, and the vessel is subject to recurring periods of pressurization,such that the vessel acts similar to an oxygen concentration system orother pressure swing absorbtion system, thereby increasing thesolubility, mass transfer, and/or uptake of gases in the vessel by themicroorganisms and liquid medium. In one embodiment, such pressure ordepressurization cycle may comprise 1-100 minutes per cycle or stage, or1-300 minutes per complete pressure-depressurization cycle. In oneembodiment, the liquid medium and/or concentration of dissolved gases inthe reactor is caused to remain relatively constant or homogenous withmixing induced by the action of the cavitation (e.g., cavitation inducedby a moving blade or liquid moving over a surface), sonication (e.g.,ultrasonication), sonic induction, gases (e.g., gas displacement),liquid displacement, mechanical pumping (e.g., rotary pump), themovement of entrained materials (e.g., the movement of liquid-entrainedplastic balls), or other means while the concentration of gases iscaused to be reduced on a proximal basis according to the cycle ofpressure in the system (e.g., from vacuum pressure to superatmosphericpressure), proximity to an absorbent material (including the associatedpressure cycle), sequential location of gas relative to gas flow path(e.g., location in gas vessels), and location or proximity to injectionport relative to exhaust port (e.g., retention time of gas). In oneembodiment, a reactor may be vertically configured, such that the heightof the vessel exceeds the width of the vessel. In one embodiment, areactor may be horizontally configured, such that the width of thevessel exceeds the height of the vessel.

In several embodiments, PHA synthesis is induced in a microorganismculture comprising methane-utilizing, heterotrophic, and/or carbondioxide-utilizing microorganisms wherein a PHA inclusion concentration(by dry biomass weight) is generated of between about 0.01% and about95%. In some embodiments, the inclusion concentration is between about25% and about 80%, including about 25 to about 35%, about 35% to about50%, about 50% to about 65%, about 65% to about 80%, and overlappingranges thereof. In some embodiments, the inclusion concentration isbetween about 0.01% and about 55%, including, about 0.01% to about 1%,about 1% to about 5%, about 5% to about 10%, about 10% to about 15%,about 15% to about 20%, about 20%, to about 25%, about 25% to about 30%,about 30% to about 35%, about 35% to about 40%, about 40% to about 45%,about 45% to about 50%, about 50% to about 55%, and overlapping rangesthereof. In some embodiments, these inclusion ratios are achieved withreduced carbon input, reduced energy expenditure, or reduced numbers ofmicroorganisms (thereby representing a more efficient generation ofPHA). In some embodiments, PHA synthesis is induced in a methanotrophic,heterotrophic, and/or autotrophic microorganism culture wherein a PHAinclusion concentration (by dry biomass weight) is generated of betweenabout 20% and about 80%, between about 30% and about 70%, between about40% and about 60%, between about 50% and about 70%, including about 50%to about 55%, about 55 to about 60%, about 60% to about 65%, about 65%to about 70%, and overlapping ranges thereof. In some embodiments of theinvention, PHA synthesis is induced in microorganism culture comprisingmethanotrophic, autotrophic, and heterotrophic microorganisms, whereinan average PHA inclusion concentration (by dry biomass weight) isgreater than about 5%, greater than about 20%, greater than about 40%,greater than about 65%, or greater than about 70% by dry cell weight.

In some embodiments, the growth culture media is manipulated to induceboth i) microorganism growth and ii) PHA synthesis within one or moreopen, non-sterile, or sterile vessels using a feast-famine cultureregime. In some embodiments, the microorganisms are subject tosuccessive alternating periods of nutrient/carbon availability andnutrient/carbon unavailability to encourage the reproductive success ofmicroorganisms that are capable of synthesizing PHA, particularly athigh inclusion concentrations. Feast-famine regimes useful for theselection of PHA producing microorganisms, including PHA-producingmethanotrophic microorganisms, over microorganisms that either cannotproduce PHA, produce PHA slowly, or produce PHA at relatively lowconcentrations are described in the art (Verlinden, et al., “Bacterialsynthesis of biodegradable polyhydroxyalkanoates,” Journal of AppliedMicrobiology, 102 (2007), p. 1437-1449, Frigon, et al., “rRNA andPoly-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast andFamine Cycles,” Applied and Environmental Microbiology, April 2006, p.2322-2330; Muller, et al., “Adaptive responses of Ralstonia eutropha tofeast and famine conditions analysed by flow cytometry,” J Biotechnol.1999 Oct. 8; 75(2-3):81-97; Reis, et al., “Production ofpolyhydroxyalkanoates by mixed microbial cultures,” Bioprocess andBiosystems Engineering, Volume 25, Number 6, 377-385, DOI:10.1007/s00449-003-0322-4.)

In some embodiments of the invention, the classical feast-famine regimeis modified to reduce PHA losses. Specifically, in the past,feast-famine regimes were thought to be effective by passing amicroorganism culture through a period wherein carbon or nutrients wereunavailable or relatively limited for metabolism, thereby forcing theculture to accumulate and/or consume intracellular PHA as a source ofcarbon to survive, and thereby selecting for microorganisms with thecapacity to synthesize and store PHA. Applicant has surprisinglydiscovered that, some microorganisms with higher concentrations ofintracellular PHA reproduce more efficiently than microorganisms withlower concentrations of intracellular PHA in periods of carbonavailability and nutrient balance. Thus, in one embodiment, a novel PHAproduction regime is employed in one or more vessel whereinmicroorganisms are subjected to two successive and recurring phases: 1)growth, wherein carbon and nutrient availability is optimized forreproduction, and 2) PHA synthesis, wherein carbon is available inexcess, and one or more nutrient is reduced or increased relative to thegrowth period to induce PHA synthesis. In some embodiments, a fractionof the vessel media is removed for downstream PHA extraction andprocessing after the PHA synthesis period, and that fraction is replacedwith lower cell density media, which simultaneously returns carbon andnutrient concentrations to reproductively favorable levels, e.g., thegrowth phase or growth conditions, and causes microorganisms to enterinto a reproductive phase without consuming significant portions ofintracellular PHA. As such, efficient PHA producing microorganismsselectively reproduce over inefficient or non-PHA producingmicroorganisms. As a result, some embodiments, i) increase the speed ofthe microorganism selection process by removing the PHA consumption steptypical to previous feast famine models and ii) reduce the loss of PHAto cellular metabolism. According to such embodiments, the feast faminemodel is converted to a feast-polymerization-feast process. In severalembodiments methanotrophic, autotrophic, and/or heterotrophic culturesare used in the feast-polymerization-feast process.

Removing a Portion of the PHA-Containing Biomass from the Culture, andExtracting PHA from the Removed PHA-Containing Biomass to ProduceIsolated PHA and PHA-Reduced Biomass

In several embodiments, following the production of a microorganismculture comprising biomass and PHA (discussed above), at least a portionof the PHA-containing biomass is removed from the culture. In severalembodiments, a portion ranging from about 20% to about 80% of thePHA-containing biomass is removed, including about 30% to about 70%,about 40% to about 60%, about 45% to about 55%, and overlapping rangesthereof. Removal of PHA-containing biomass may be performed by a numberof methods, including centrifugation, filtration, density separation,flocculation, agglomeration, spray drying, or other separationtechnique. In some embodiments, dewatering (e.g., by centrifugation)results in a biomass having a desirable water content that facilitatesdownstream processing of the biomass. For example, in some embodiments,centrifugation of the PHA-containing biomass reduces the amount ofculture media (increases the relative biomass concentration) to aconcentration range between about 100 and about 500 grams of biomass perliter of culture media. In some embodiments, the concentration is of thebiomass is adjusted to about 100 to about 200 g/L, about 200 to about300 g/L, about 300 to about 400 g/L, about 400 to about 500 g/L, andoverlapping ranges thereof. Advantageously, such an approach alsoproduces, as an effective by-product, clarified culture media that canbe optionally treated, measured, or recycled into one or more culturevessels.

In some embodiments, after a portion of the PHA-containing biomass isremoved from the culture, PHA is extracted from the removedPHA-containing biomass to produce isolated PHA and PHA-reduced biomass.In some embodiments, supercritical (SC) fluids, such as SC-CO₂ orSC-water are used to purify PHA, such that proteins and/or non-PHAmaterials are rendered at least partially solubilized in SC-CO₂,SC-water, high temperature or high pressure water, and/or mixturesthereof. In some embodiments, compatibilizing extraction agents may beused, such as non-PHA polymers that maintain miscibiluty with PHA andhigh solubility in SC-fluids, such that the PHA, miscible polymer, andSC-fluid produce a low viscosity solution capable of separating PHA fromnon-PHA material.

As used herein, the terms “extraction” and “PHA extraction” shall begiven their ordinary meaning and shall be used interchangeably todescribe the removal and/or separation of PHA from biomass. PHAs may beextracted from biomass by several processes, including, but not limitedto, the use of chemicals (e.g., solvents) alone or in combination withmechanical means and/or enzymes. These processes include the use of:solvents, such as acetone, ethanol, methanol, methylene chloride,dichloroethane, with and/or without the application of pressure and/orelevated temperatures, supercritical carbon dioxide, enzymes, such asproteases, surfactants, pH adjustment, including the protonic orhydroxide-based dissolution of non-PHA biomass, and/or hypochlorite (oranother solvent) to dissolve non-PHA biomass, including the use ofhypochlorite in conjunction with another solvent, such as methylenechloride or with, carbon dioxide, enzymes, acids, bases, polymers, orsurfactants, or combinations thereof. In some embodiments of theinvention, PHA is extracted by solvent extraction from a PHA-containingbiomass comprising gas-utilizing microorganisms and/or biomass-utilizingmicroorganisms to produce isolated PHA and PHA-reduced biomass. In somesolvent-based embodiments, solvents suitable for dissolving the PHA areused, including carbon dioxide, acetone, methylene chloride, chloroform,water, ethanol, and methanol. In some embodiments, particular ratios ofsolvent to PHA provide optimal dissolution of PHA from the culture, andtherefore lead to improved extraction and isolation efficiency andyield. For example, in some embodiments, ratios of solvent to PHA (ingrams) of about 500:1 are used. In some embodiments, ratios of about0.01:1 are used. In some embodiments, ratios ranging from between about500:1 and about 0.01:1 are used, such as about 0.05:1, about 1.0:1,about 1.5:1, about 20:1, about 250:1, about 300:1, about 350:1, about400:1, or about 450:1.

As discussed above, changes in temperature and/or pressure may also beused to facilitate the extraction of PHA from the PHA-containingbiomass. In some embodiments, the extraction solvent chosen dictates thelimits of temperature, pressure, and/or incubation times that are used.In some embodiments, solvent is combined with PHA-containing biomass andincubated for several minutes up to several hours. For example, in someembodiments, incubation is for about 10 minutes, while in otherembodiments, overnight incubation times are used. In some embodiments,incubation times range from 30 minutes to about 1 hour, about 1 hour toabout 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours,and from about 10 hours to overnight. Choice of incubation time isdetermined by solvent, culture density (e.g., number of microorganisms),type of organisms, expected PHA yield, and other similar factors.

Incubation temperature is also tailored to the characteristics of agiven culture. Incubation temperatures can range from below roomtemperature to elevated temperatures of up to about 150° C. or about200° C. For example, depending the solvent and other variables of theculture, temperatures are used that range from about 10° C. to 25° C.,from about 25° C. to about 40° C., from about 40° C. to about 55° C.,from about 55° C. to about 60° C., from about 60° C. to about 75° C.,from about 75° C. to about 90° C., from about 90° C. to about 105° C.,from about 105° C. to about 120° C., from about 120° C. to about 135°C., from about 135° C. to about 150° C., from about 150° C. to about200° C., and overlapping ranges thereof.

As can be appreciated, if changes in temperature are made to a culturein a closed vessel, changes in pressure result. In some embodiments,increased pressure provides a shearing effect that aids in theliberation of PHA from the microorganisms. In some embodiments, pressureis regulated within a particular range. For example, in someembodiments, pressure of the reaction of the PHA-containing biomass withsolvent occurs between about 40 and 30,000 psi, including about 50 toabout 60 psi, about 60 to about 70 psi, about 70 to about 80 psi, about80 to about 90 psi, about 90 to about 100 psi, about 100 to about 125psi, about 125 to about 150 psi, about 150 to about 175 psi, about 175to about 200 psi, about 200 to about 1000 psi, about 1000 to about 5000psi, about 5000 psi to about 10,000 psi, about 10,000 to about 20,000psi, about 20,000 to about 30,000 psi, and overlapping ranges thereof.Additional sources of shear (e.g., agitation, pumping, stirring etc.)are optionally used in some embodiments to enhance the extraction ofPHA. Any one, or combination, of the PHA extraction methods describedherein, or disclosed in the art, may be utilized as a method to carryout PHA extraction and remove PHA from the PHA-containing biomass.

In several embodiments, a solvent-based extraction system is utilized tocarry out PHA extraction. In some embodiments, solvents are utilized tocarry out PHA extraction at high temperatures, wherein PHA extractionoccurs simultaneous with a temperature-enhanced breakdown or dissolutionof PHA-containing biomass. In some embodiments, one or more solvent isutilized that is biodegradable and metabolically assimilable by theculture, such that PHA-reduced biomass comprising biomass and one ormore biodegradable solvent may be contacted with the culture, and boththe PHA-reduced biomass and the solvent may be utilized by the cultureas a source of carbon. Non-limiting examples of such solvents includecarbon dioxide, acetone, ethanol, methanol, and methylene chloride,among others.

In several embodiments a mixture of solvent and PHA comprises multiplephases, e.g. an aqueous phase and an organic phase. In some embodiments,solvent-based extraction comprises a more uniform mixture of solvent andPHA. In some embodiments, depending on the solvent the phases areseparated prior to recovery of the PHA. In some embodiments, a non-PHApolymer is also used, alone or in conjunction with other processes, forseparation, flocculation, or other processing. In some embodiments,centrifugation is employed to further distinguish and separate thephases of the mixture (e.g., separation of the solvent-PHA phase fromthe water-biomass phase). In some embodiments, heat is also employed tomaintain the solubility of the PHA in a given solvent.

Depending on the embodiment, the solubility of PHA varies with thesolvent used, and therefore the temperature (if adjusted) and theseparation techniques are tailored to match the characteristics of agiven solvent. Thus, in some embodiments employing centrifugation, forexample, a low speed centrifugation is used to separate the solvent-PHAphase from the water-biomass phase. In other embodiments, depending onthe solvent, higher speed centrifugation is used. In some embodiments,centrifugation is employed in stages, e.g., low speed centrifugationfollowed by high speed centrifugation. Any of a variety of centrifugescan be employed, depending on the solvent used, for example, basketcentrifuges, swinging bucket centrifuges, fixed rotor centrifuges,disc-back centrifuges, supercentrifuges, or ultracentrifuges.

In some embodiments, adjustable discharge ports suitable for aparticular centrifuge are used in order to control the rate and degreeof separation of solvent-PHA phase from the water-biomass phase. In someembodiments, the concentration of water in the water-biomass phase isadjusted to allow for suitable flow of the mixture through thecentrifuge (or within a centrifuge tube). For example, in someembodiments, flow is suitable for separating the phases when theconcentration of biomass (relative to water) is between about 1 and 100g/L. In some embodiments, the concentration is between about 10 to about20 g/L, about 20 to about 30 g/L, about 30 to about 40 g/L, about 40 toabout 50 g/L, about 50 to about 60 g/L, about 60 to about 70 g/L, about70 to about 80 g/L, about 80 to about 90 g/L, about 90 to about 100 g/L,about 100 to about 200 g/L, about 200 to about 400 g/L, about 400 toabout 600 g/L, and overlapping ranges thereof.

In still additional embodiments, increases in temperature not onlyfacilitate the extraction of the PHA, they also facilitate the isolationof the PHA from the solvent (e.g. increased temperature increasessolvent evaporation).

In some embodiments, an extraction process is carried out to remove PHAfrom a microorganism in such a manner that the microorganism isdeactivated. In some embodiments, the deactivation is permanent, whilein some embodiments the deactivation is temporary. Without being boundby theory, it is believed that PHA extraction techniques which do notpermanently disable microorganisms enable the PHA-reduced biomassgenerated thereby to contribute to the metabolism of carbon sourcesafter a PHA extraction process, including through intracellular andextracellular metabolism. In one embodiment, methods useful for thetemporary disablement of microorganisms include solvent extraction,including solvent extraction carried out below about 100° C., andparticularly at intracellular temperatures below about 100° C.,including extraction temperatures of about 10° C. to about 30° C., about30° C. to about 50° C., about 50° C. to about 60° C., about 60° C. toabout 70° C., about 70° C. to about 80° C., about 80° C. to about 90°C., about 90° C. to about 100° C., and overlapping ranges thereof.

In several embodiments, the PHA concentration of PHA-containing biomassis reduced as a result of the PHA extraction process. In severalembodiments, the PHA concentration of PHA-containing biomass is reducedby at least about 0.01% (by dry cell weight). In some embodiments, thePHA concentration is reduced by about 10% to about 50%, about 50% toabout 70%, about 70% to about 75%, about 75% to about 80%, about 80% toabout 85%, about 85%, to about 90%, about 90% to about 95%, about 95% toabout 99.9%, and overlapping ranges thereof.

While a variety of methods are known to enable the extraction PHA frombiomass, most methods can be categorized into one of two classes: a)solvent-based extraction, or b) NPCM (non-polymer cellular material)dissolution-based extraction. NPCM dissolution-based extraction methodsutilize chemicals (such as hypochlorite, or bleach), enzymes (such asprotease), heat (especially to reach temperatures above 100° C.), pH(acids and bases), and/or mechanical means (such as homogenization) tobreak down, oxidize, and/or emulsify non-PHA cellular material. In somecases, extraction methods from both categories can be combined, such asthe simultaneous utilization of hypochlorite and methylene chloride.

NPCM dissolution-based extraction methods require continuous andnon-recoverable chemical inputs, such as hypochlorite, peroxides,enzymes, and pH adjustors, and also generate significant waste disposalissues. Thus, while these methods are effective, the use ofsolvent-based extraction methods is generally preferred in the industrydue to the capacity of solvents to be distilled and recovered forcontinuous re-utilization in a closed loop cycle. Unfortunately, despiteits benefits, some solvent-based extraction methods are energy intensiveprocesses that play a major role in the high cost of PHA production,often accounting for more than 50% of total production costs.Accordingly, there exists a significant need for a novel method tosignificant increase the energy efficiency of solvent-based extraction.

In several embodiments, a process for the extraction ofpolyhydroxyalkanoates from biomass using a solvent-based extractionmethod is provided, wherein the energy required to carry out the processis reduced relative to prior solvent-based extraction methods.Specifically, in one embodiment a high efficiency PHA extraction processis provided comprising providing a PHA-containing biomass comprising PHAand water, mixing the biomass with a solvent at a temperature sufficientto dissolve at least a portion of the PHA into the solvent and at apressure sufficient to enable substantially all or part of the solventto remain in liquid phase, thereby producing a PHA-lean biomass phaseand a PHA-rich solvent phase comprising solvent, water, and PHA,separating the PHA-rich solvent phase from the PHA-lean biomass phase ata temperature and pressure sufficient to enable substantially all orpart of the solvent to remain in the liquid phase and preventsubstantially all or part of the PHA within the PHA-rich solvent phasefrom precipitating, reducing the pressure or increasing the temperatureof the PHA-rich solvent phase to cause the solvent to vaporize and thePHA to precipitate or become a solid while maintaining the temperatureand/or the pressure of the PHA-rich solvent phase to prevent all or partof the temperature-dependent precipitation of the PHA into water, andcollecting the solid PHA material, including optionally separating theprecipitated PHA from the solvent and/or the water.

In the past, PHA precipitation has been induced in PHA-rich solvent bya) adding a non-PHA solvent to the solvent phase to reduce thesolubility of PHA in the solvent phase and/or b) reducing thetemperature of the solvent phase to reduce the solubility of PHA in thesolvent. In particular, some methods 1) dissolve PHA in a solvent byincreasing the temperature of the solvent and 2) precipitate PHA byreducing the temperature (and, thus, solubility) of the solvent. Othermethods require adding water to a solution of PHA-rich solventcomprising dissolved PHA, wherein the addition of water to the solutionreduces the solubility of the PHA in the solvent and causes the PHA toprecipitate into the solvent and/or water. (For example, U.S. Pat. Nos.4,562,245; 4,968,611; 5,894,062; 4,101,533, all herein incorporated byreference.) In each of these cases, energy efficiency is compromised;specifically, by adding water or a non-PHA solvent to reduce the PHAsolubility of a solvent, additional energy is required for downstreamwater/non-solvent removal, heating, and/or distillation. By reducing thetemperature of the solvent to reduce the solubility of the solvent andinduce PHA precipitation, heat energy is redundantly expended, as thesolvent must be re-heated for distillation and recovery. Therefore, inseveral embodiments, rather than adding a non-solvent to a PHA-solventor reducing the temperature of the PHA-solvent to effect PHAprecipitation, pressure and/or an increase in temperature is used toinduce the precipitation or solidification of the PHA withoutredundantly reducing the temperature of solvent. Thus, in suchembodiments, there is a significant reduction in the energy required toheat and/or distill non-solvent and/or solvent in downstream PHAprocessing.

In one embodiment, the extraction process is substantially carried outat intracellular temperatures less than about 100° C. In otherembodiments, temperatures for extraction range from about 10° C. toabout 30° C., from about 30° C. to about 50° C., from about 50° C. toabout 70° C., from about 70° C. to about 90° C., from about 90° C. toabout 120° C., from about 100° C. to about 140° C., from about 20° C. toabout 150° C., or from about 120° C. to about 180° C., or higher. In oneembodiment, cells are reused for polymerization following the extractionprocess as viable cells. In one embodiment, PHA-containing biomass istreated to one or more chemical treatment steps to control, modify, orincrease the concentration or functional characteristics (e.g.,molecular weight, monomer composition, melt flow profile, purity,non-PHA residuals concentration, protein concentration, DNAconcentration, antibody concentration, antioxidant concentration) of PHAin a PHA-containing material or biomass. In one embodiment, temperatureis used to control, modify, reduce, or optimize the molecular weight,polydispersity, melt flow, and other characteristics of PHA. In oneembodiment, temperature and/or time is used to control the molecularweight of PHA between the range of 5,000,000 and 10,000 Daltons. In oneembodiment, a slurry comprising PHA-containing biomass and a culturemedia is subject to one or more water removal steps or water additionsteps to increase the concentration of PHA in a PHA-containing biomass.In one embodiment, the water removal step is a dewatering step orcombination of dewatering steps, such as centrifugation, filtration,spray drying, flash drying, and/or chemical dewatering (e.g., withacetone, ethanol, or methanol), wherein at least a portion of the waterconcentration relative to the concentration of PHA-containing biomass inthe slurry is reduced. In one embodiment, a temperature and/or pressurecontrol step is carried out under atmospheric (0 psi), sub-atmospheric(−100-0 psi), or above-atmospheric pressure (e.g., 0-30,000 psi) and attemperature conditions wherein the PHA-containing biomass, or the liquidin and/or around the PHA-containing biomass, is maintained, for at leasta period of time, at a temperature ranging from about −30 to about 10degrees Celsius, about 10 degrees Celsius to about 100 degrees Celsius,about 10 degrees Celsius to about 150 degrees Celsius, about 20 degreesCelsius to about 250 degrees Celsius, and/or about 100 to about 200degrees Celsius. In one embodiment, the PHA-containing biomass issubject to a dewatering step before or after the temperature and/orpressure control step, wherein the dewatering step is centrifugation,filtration, and/or spray drying, to produce a fully or partiallyde-watered PHA-containing biomass or PHA-containing biomass slurry,wherein the water concentration of the dried slurry is less than about99%, less than about 95%, less than about 80%, less than about 60%, lessthan about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, less than about 3%, less than about 2%,or less than about 1% water. In one embodiment, the PHA-containingbiomass is subject to a temperature control step, wherein the liquidchemicals within and/or around the biomass, e.g., water, methylenechloride, carbon dioxide, and/or ammonia, is controlled and maintainedat a temperature of at least −30, at least −10, at least −5, at least−4, at least −3, at least −2, at least −1, at least 0, at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 110, at least 120,at least 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 210, at least 220, atleast 230, at least 240, at least 250, at least 260, at least 270, atleast 280, at least 290, at least 300 degrees Celsius (or overlappingranges of those temperatures), wherein the solubility, dispersitivity,or homogeneity of non-PHA material increases in the liquid in and/oraround the biomass. In one embodiment, the PHA-biomass is not driedprior to such temperature control step. In one embodiment, thePHA-containing biomass is dried or de-watered prior to such temperaturecontrol step. In one embodiment, the PHA-containing biomass is filteredor centrifuged following the temperature control step. In oneembodiment, the PHA-containing cell slurry is not dewatered, forexample, by centrifugation or other drying mechanism, prior to thetemperature control step. In one embodiment, a mechanism to impart shearonto or into the PHA-containing biomass is coupled with a temperaturecontrol step; such shear may be imparted in the form of one or moreshear induction mechanisms, e.g., centrifugal pump, agitator, blender,high shear mixer, vortex mixer, etc. In one embodiment, thePHA-containing biomass is dewatered in one step and the treatedPHA-containing biomass is further dewatered in one or more additionalsteps. In one embodiment, the PHA-containing biomass is dewatered, waterand/or other chemicals are added and temperature and/or pressure iscontrolled, and the treated PHA-containing biomass is further dewateredand/or purified. In one embodiment, the water and/or chemicals within,around, and/or added to the PHA-containing biomass is temperature and/orpressure controlled, and the treated PHA-containing biomass is furtherpurified in one or more purification steps. In one embodiment, thetemperature control step process time is approximately 1 second,approximately 5 seconds, approximately 10 seconds, approximately 25seconds, approximately 60 seconds, approximately 2 minutes,approximately 5 minutes, approximately 20 minutes, approximately 45minutes, approximately 1 hour, approximately 2 hours, approximately 5hours, approximately 6 hours, approximately 7 hours, approximately 12hours, approximately 15 hours, approximately 24 hours, approximately 36hours, approximately 48 hours, or overlapping ranges of those times. Inone embodiment, inorganic materials may be used to effect PHAmodification, purification, or extraction, including carbon dioxide anddinitrogen. In one embodiment, the PHA-containing slurry or biomass istreated with carbon dioxide under elevated temperatures and pressures,including supercritical ranges, to induce PHA extraction or functionalmodification of PHA. In one embodiment, organic solvents, includingmethylene chloride, acetone, chloroform, dichloroethane, ethanol, and/ormethanol are used in conjunction with any of the above steps. In oneembodiment, solvents or extraction materials may be used or recycled forbiomass production, biogas production, and/or PHA synthesis.

In one embodiment, chemicals are added to a PHA-containing biomass tocause the crystallization of PHA. In one embodiment, methylene chloride,carbon dioxide, acetone, water, dichloroethane, or methanol may be addedto a PHA-containing biomass in order to induce the crystallization ofPHA in the PHA-containing biomass. In some embodiments, this step may beuseful for the downstream processing of PHA, wherein crystallized PHA isless prone than amorphous to degradation, including molecular weightloss, when contacted with extraction chemicals, including solvents,enzymes, acids, bases, and bleach. In one embodiment, silicon, silica,derivatives thereof, and/or chemicals containing silicon may be added tothe PHA-containing biomass in order to impact the metabolic status ofthe culture, and thereby control the functional characteristics of thePHA produced by the culture, including molecular weight, monomercomposition, co-polymer structure, melt index, and polydispersity.

The removal of non-PHA materials from PHA often accounts for asignificant fraction of PHA production costs. As a specific example,pigments often cause unwanted discoloration of PHA, and must be removedthrough costly processes, such as ozonation, peroxide washing, acetonewashing, ethanol washing, solvent refluxing, hypochlorite digestion,enzymatic degradation, surfactant dissolution, or other methodsdisclosed in the art. In several embodiments, a non-sterile process isused to select for microorganisms exhibiting minimal pigmentation.Applicant has surprisingly discovered that, by manipulating theconcentration of dissolved oxygen in a microorganism system, a culturemay be selected wherein white, tan, off-white, light brown, and/or lightyellow pigments are exhibited rather than purple, red, pink, dark brown,orange, or other heavy pigments. Specifically, in some embodiments, anexcess of dissolved oxygen is introduced into a growth media oversuccessive periods, resulting in selective growth of strains ofmethanotrophic microorganisms which produce white, tan, off-white, lightbrown, and light yellow pigments, rather than those producing pink, red,purple, dark yellow, dark brown, and/or other heavy pigments. As aresult, such embodiments, reduce the need for costly downstreampigmentation removal.

As used herein, the term “PHA-reduced biomass” or “substantiallyPHA-reduced biomass” shall be given its ordinary meaning and shall beused to describe a biomass material wherein the concentration of PHArelative to non-PHA material has been reduced in a PHA-containingbiomass through the utilization of a PHA extraction process. In someembodiments, PHA-reduced biomass is further treated in a variety ofways. In some embodiments, the further treatment includes, but is notlimited to, one or more of dewatering, chemical treatment, sonication,additional PHA extraction, homogenization, heat treatment, pH treatment,hypochlorite treatment, microwave treatment, microbiological treatment,including both aerobic and anaerobic digestion, solvent treatment, waterwashing, solvent washing, and/or drying, including simple or fractionaldistillation, spray drying, freeze drying, rotary drying, and/or ovendrying.

In one embodiment, PHA-reduced biomass is substantially dried, whereinthe resulting dried material comprises less than about 99% liquids,including water, solvents, salts, and/or growth-culture media. In someembodiments, the drying processes disclosed herein yield a driedmaterial containing between about 95% and about 75% liquids, betweenabout 75% and about 50% liquids, between about 50% and about 25%liquids, between about 25% and about 15% liquids. between about 15% andabout 10% liquids, between about 10% and about 1% liquids, andoverlapping ranges thereof. In some embodiments, drying is complete(e.g., between about 1% and 0.1% liquids, or less). In anotherembodiment of the invention, a liquid phase comprising PHA-reducedbiomass is subjected to filtration, centrifugation, densitydifferentiation, or other method to increase the solids content of thePHA-reduced biomass.

Traditionally, the separation of biomass from liquid growth media isdifficult and impractical due to the plugging and foulingcharacteristics of biomass. In several embodiments, a method enablingthe efficient filtration of microorganisms is provided. In someembodiments, a liquid chemical is added to the growth media comprisingmicroorganisms, wherein the liquid chemical is ethanol, acetone,methanol, methylene chloride, ketones, alcohols, and/or chlorinatedsolvents, or a combination thereof. In some embodiments, microorganismsare then efficiently separated from liquid growth media using standardfiltration equipment, such as a Buchner filter, filter press, or similarapparatus. In one embodiment, approximately 2 parts acetone are mixedwith one part water, including both intracellular and extracellularwater, to effect the efficient filtration of microorganisms comprisingthe water.

As used herein, the terms “isolated PHA” and “substantially isolatedPHA” shall be given their ordinary meaning and shall refer to PHA thathas been removed from a biomass material as a result of an extractionprocess, or a biomass material wherein the concentration of PHA relativeto non-PHA material has been increased by an extraction process. Inseveral embodiments, isolated PHA is further treated in one or more of avariety of ways, including, but not limited to, purification,filtration, washing, oxidation, odor removal, pigment removal, lipidremoval, non-PHA material removal, and/or drying, includingcentrifugation, filtration, spray drying, freeze drying, simple orfractional distillation, or density differentiation. Methods for thepurification of PHA include the use of peroxides, water, hypochlorite,solvents, ketones, alcohols, and various other agents to separate andremove non-PHA material from PHA material. In some embodiments, PHA isremoved from a microorganism culture by solvent extraction to produceisolated PHA in a PHA-rich solvent phase and PHA-reduced biomass in aPHA-lean liquid phase. In some embodiments, the solvent phase isseparated from the liquid phase by filtration or centrifugation. In someembodiments, both centrifugation and filtration are used in combination(e.g., sequentially). In some embodiments, centrifugation is optionallyfollowed by filtration. In other embodiments, filtration is optionallyfollowed by centrifugation. Filtration, in some embodiments is performedunder vacuum pressure, via gravity feed, under positive pressure, or inspecialized filtration centrifuges. In some embodiments, the filter poresize is adjusted based on the species composition of the microorganismculture. In some embodiments, pore sizes of up to 200 μm are used. Insome embodiments, smaller pore sizes are used, for example 15 to 20 μm,10 to 15 μm, 5 to 10 μm, 1 to 5 μm, 0.001 to 1 μm, and overlappingranges thereof.

In addition to the steps outlined above, additional steps may be takento remove solvent from the extracted PHA, including evaporation, solventcasting, steam stripping, heat treatment, and vacuum treatment, each ofwhich may be preferential, cost-effective, time-effective, oradvantageous depending on the volatility and type of solvent used. Inother embodiments, active processes can be used to reduce the solventcontent of the solvent-PHA mixture. For example, in certain embodiments,alterations in temperature of certain solvents change the solubility ofthe PHA in the solvent, which effectively removes solvent from the PHA(e.g., the solvent is now separable from a precipitated PHA). In someembodiments, filtration, solvent temperatures, or vacuum treatment canbe increased to reduce a portion of the solvent. In some embodiments,solvent to PHA ratios post extraction, filtration, evaporation, solventcasting, steam stripping, heat treatment, and/or vacuum treatment rangefrom about 0.1:1 to about 1,000:1, including about 0.2:1, about 0.3:1,about 4.0:1, about 5.0:1, about 10.0:1, about 20.0:1, about 60:1, about70:1, about 80:1, about 90:1, about 100:1, about 200:1, about 500:1, andabout 900:1.

As a result of the processes disclosed above, in some embodiments, thesolvent is substantially removed from the isolated PHA in the PHA-richsolvent phase and the liquid is substantially removed from thePHA-reduced biomass in the PHA-lean liquid phase. In some embodiments,the isolated PHA is dried in a heated vessel to produce substantiallypure isolated PHA (e.g., at least 80% PHA by dry weight, preferably atleast 98% PHA by weight, more preferably at least 99% PHA by weight).

Numerous varieties of heated or drying vessels may be used to dry theisolated PHA, including ovens, centrifugal dryers, air dryers, spraydryers, and freeze dryers, among others. In some embodiments, heat isapplied to a drying vessel to speed the process and/or to remove (e.g.,evaporate traces of solvent from the PHA). The moisture content of theisolated PHA will depend, in some embodiments, on the solvent used, andthe corresponding separation technique used (as described above). Forexample, a volatile solvent in combination with ultracentrifugationwould result in a less moist extracted PHA, while a less activeseparation technique (e.g., gravity phase separation) would yield a moremoist extracted PHA. In some embodiments, internal dryer temperaturesrange from 20° C. to 40° C. to about 200° C. In some embodiments,internal temperatures range from about 50° C. to about 90° C., about 90°C. to about 180° C., about 65° C. to about 175° C. and overlappingranges thereof. In some embodiments, outlet temperatures aresubstantially lower than inlet on internal temperatures. In someembodiments, outlet temperatures range from 30° C. to 90° C. In someembodiments, outlet temperatures are between about 35° C. to 40° C.,about 40° C. to about 45° C., about 45° C. to about 50° C., about 50° C.to about 55° C., about 55° C. to about 90° C., and overlapping rangesthereof. It shall also be appreciated that the internal and outlettemperatures may be adjusted throughout the drying process (e.g., thetemperature difference may initially be large, but decrease over time,or vice versa).

Depending on the embodiment, the type of dryer used, and thetemperatures used (if other than atmospheric temperatures) are easilytailored to correspond to the techniques used in the extraction process.In some embodiments, particular dryer components are beneficial in theisolation of PHA. For example, depending on the moisture content of theextracted PHA (e.g., the amount of residual solvent) particularcomponents of an evaporative-type dryer, such as an oven dryer, rotarydryer, spin flash dryer, conveyor dryer, spray dryer (equipped withvarious types of nozzle types, including rotary atomizor, single flowatomizer, mist atomizer, pressure atomizer, dual-flow atomizer)convection heat dryer, tray dryer, scrape-flash dryer, or other dryertype are used. By way of additional example, if a freeze dryer (e.g., alyophilizer) is used, in some embodiments a manifold dryer is used,optionally in conjunction with a heat source. Also by way of example, atray lyophilizer can be used, in some embodiments, with the isolated anddried PHA being stored and sealed in containers (e.g., vials) beforere-exposure to the atmosphere. In certain embodiments, such an approachis used when long-term storage of the PHA is desired.

It shall also be appreciated that certain varieties of heated/dryingapparatuses have adjustable flow rates that can be tailored to themoisture content of the isolated PHA. For example, an isolated PHAhaving a high moisture content would be fed into a dryer at a slowerinput rate to allow a higher degree of drying per unit of PHA inputtedinto the dryer. Conversely, a low moisture content isolated PHA wouldlikely require less time to dry, and therefore could be input at afaster rate. In some embodiments, input rates of isolated PHA range fromseveral hundred liters of isolated PHA-solvent mixture per minute downto several milliliters per minute. For example, input rates can rangefrom about 10 mL/min to about 6 L/min, including about 10 ml/min toabout 50 ml/min, about 50 mL/min to about 100 ml/min, about 100 ml/minto about 500 ml/min, about 500 ml/min to about 1 L/min, about 1 L/min toabout 2 L/min, about 2 L/min to about 4 L/min, about 4 L/min to about 6L/min, and about 100 L/min to about 500 L/min.

A range of PHA functional characteristics can be attained by mixing onePHA molecule, such as PHB, with various PHA polymers, including PHB, atvarious molecular weights. Therefore, in several embodiments, a firstisolated PHA is heated to reduce the molecular weight of the firstisolated PHA, and then subsequently mixed with a second PHA wherein themolecular weight of the second PHA is higher than the molecular weightof the first PHA. With such embodiments, Applicant has surprisinglydiscovered methods to functionalize one or more PHAs, including PHB. Inadditional embodiments of the invention, the molecular weight of a firstPHA is reduced from about 800,000 to about 5,000,000 Daltons to about30,000 to about 800,000 Daltons and mixed with a second PHA with amolecular weight of about 800,000 to about 5,000,000 Daltons to modifythe functionalities of the input PHAs. In yet another embodiment, afirst PHA is mixed with a second PHA wherein the molecular weight of thefirst PHA is at least 0.1% less than the molecular weight of the secondPHA. In some embodiments, the difference in molecular weight between thefirst and second PHA is about 0.1% to about 1%, about 1% to about 10%,about 10% to about 20%, about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,and overlapping ranges thereof. In still additional embodiments, PHAshaving greater differences in molecular weight are used. In yet anotherembodiment, the molecular weight of a first PHB is reduced to less thanabout 100,000-500,000 Daltons and mixed with a second PHA with amolecular weight greater than about 100,000-500,000 Daltons to modifythe functionality of the input PHB. Input PHA and PHB weight may varyfrom the ranges disclosed above, but based on the differences in themolecular weights, the alteration in functionality of the input PHB isstill achieved.

In several embodiments, the molecular weight is adjusted in order totune, alter, or otherwise modify one or more characteristics of theend-product PHA produced. For example, in several embodiments, reductionof the molecular weight of the PHA, in turn, reduces the crystallinityof the PHA. In several embodiments, the molecular weight of a PHA isreduced, such that i) the crystallinity of the PHA is also reducedand/or ii) the onset or rate of crystallization is caused to slow. Inseveral embodiments, this is advantageous because the reducedcrystallinity allows use of the PHA in products with reduced brittleness(e.g., increased flexibility and/or durability). In several embodiments,the reduced rate of crystallization increases the ease of manufacturingand reduces associated costs with maintaining PHA in a non-crystallizedstate. In several embodiments the rate of crystallization is adjusted(via molecular weight variation) such that crystallization occurs withina period of time ranging from about 1 second to about 60 seconds, about10 seconds to about 2 minutes, about 3 minutes to about 10 minutes,about 1 minute to about 60 minutes, about 10 minutes to 3 hours, about 1hour to about 12 hours, about 6 hours to about 24 hours, about 18 hoursto about 3 days, about 1 day to about 10 days, about 3 days to about 30days, about 10 days to about 90 days, about 90 days to about 180 days,or more than 365 days (and overlapping time ranges therebetweeen).

In one embodiment, the molecular weight of a PHA is reduced by about10%, about 20%, about 50%, about 75%, or about 99%. In severalembodiments, the starting molecular weight is in a range from about100,000 to about 3,000,000 daltons. In several embodiments,post-reduction, the molecular weight ranges from about 50 to about200,000 daltons, from about 50 to about 50,000 daltons, from about 50 toabout 120,000 daltons, from about 50 to about 140,000 daltons, fromabout 50 to about 160,000 daltons, from about 50 to about 180,000daltons, from about 500 to about 200,000 daltons, from about 1000 toabout 200,000 daltons, from about 5000 to about 200,000 daltons, fromabout 10,000 to about 200,000 daltons, from about 20,000 to about200,000 daltons, from about 50,000 to about 200,000 daltons, andoverlapping ranges thereof. In one embodiment, the molecular weight of aPHA is reduced from an initial range of about 250,000 to about 1,800,000daltons to a range of about 20,000 to about 150,000 daltons.

In several embodiments, upon heating such reduced-MW PHA to its meltingpoint or above its melting point, and subsequently cooling the reduce-MWPHA to below its melting point, the crystallinity of the resultant PHAis reduced relative to the crystallinity of non-reduced molecular weightmolecular weight PHA subjected to the same conditions. In severalembodiments, the reduction in crystallinity of the reduced-MW PHA is byover about 10%, about 15%, about 25%, about 50%, about 75%, or about 90%relative to the crystallinity of non-reduced MW PHA. In severalembodiments, such crystallinity PHAs having reduced crystallinity mayoptionally include one or more of PHB, PHBV, PHHX, PHV, PHO, and/or arange of other PHAs.

In one embodiment, the invention comprises a PHA comprising carbonderived from PHA-reduced biomass, wherein the PHA-reduced biomasscomprises carbon derived from one or more carbon-containing gas and/orone or more source of source of carbon. In one embodiment, PHA isco-mingled and/or melted with biomass, including, as examples,methanotrophic, autotrophic, heterotrophic biomass, and/or PHA-reducedbiomass, to improve the functional characteristics of the PHA. In oneembodiment, PHA is co-mixed and/or melted with biomass, including, asexamples, methanotrophic, autotrophic, heterotrophic biomass, and/orPHA-reduced biomass, to improve the functional characteristics of PHA.In one embodiment, the percentage of non-PHA microorganism biomassincluded in a PHA, PHA compound, or PHA mixture is about 0.00001% toabout 0.001%, about 0.001% to about 0.01%, about 0.01% to about 0.1%, toabout 0.1% to about 0.5%, about 0.5% to about 1%, about 1%, to about 2%,about 2% to about 3%, about 3% to about 5%, about 5% to about 7%, about7% to about 10%, about 10% to about 15%, about 15% to about 20%, about20% to about 30%, about 30% to about 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 98%, about 98% to about 99.99%, andoverlapping ranges thereof. In some embodiments, the inclusion ofmicroorganism biomass to a PHA improves the functional characteristicsof a PHA by acting as one or more of the following: nucleating agent,plasticizer, compatibilizer, melt flow modifier, mold release agent,filler, strength modifier, elasticity modifier, or density modifier. Insome embodiments, microscopic size, molecular weight, molecular weightdispersity, and/or chemical nature of microorganism biomass and/ormodified, including modified or non-modified nucleic acids and proteins,including biomass that has been subject to a modification step, isparticularly and surprisingly effective as a functionalization agent forPHA. In some embodiments, biomass or modified biomass acts as asurprisingly effective compatibilizer and functional modifier for PHAand for PHA and non-PHA polymers, such as polypropylene andpolyethylene. In one embodiment, biomass and/or modified biomass ismixed with a PHA material, including PHA and/or other non-PHA polymers,to modify the nucleation, plasticization, compatibilization, melt flow,density, strength, elongation, elasticity, mold strength, mold release,and/or bulk density characteristics of a PHA material, which may bemelted, extruded, film blown, die cast, pressed, injection molded, orotherwise processed. In one embodiment, PHA is partially or not removedfrom PHA-containing microorganism biomass prior to melt processing,e.g., extrusion, injection molding, etc. In one embodiment, non-PHAbiomass parts or materials are caused to remain with PHA derived from aPHA-containing biomass in order to modify or control the functionalcharacteristics of a PHA material. In one embodiment, PHA is present inPHA-containing biomass at a concentration ranging from 1-99.99999%,1-99.99%, 1-99%, 5-99%, 10-99%, 20-99%, 30-99%, 50-99%, 70-99%, 80-99%,90-99%, 95-99%, or 98-99%, and overlapping ranges thereof. In oneembodiment, PHA, biomass, and one or more non-PHA material, polymer, orthermoplastic are mixed, melted, or processed together. In oneembodiment, the non-PHA polymer consists of one or more of thefollowing: polypropylene, polyethylene, polystyrene, polycarbonate,acrylonitrile butadiene styrene, polyethylene terephthalate, polyvinylchloride, fluoropolymers, liquid crystal polymers, acrylic,polyamide/imide, polyarylate, acetal, polyetherimide, polyetherketone,nylon, polyphenylene sulfide, polysulfone, cellulosics, polyester,polyurethane, polyphenylene oxide, polyphenylene ether, styreneacrylonitrile, styrene maleic anhydride, thermoplastic elastomer, ultrahigh molecular weight polyethylene, epoxy, melamine molding compound,phenolic, unsaturated polyester, polyurethane isocyanates, urea moldingcompound, vinyl ester, polyetheretherketone, polyoxymethylene plastic,polyphenylene sulfide, polyetherketone, polysulphone, polybutyleneterephthalate, polyacrylic acid, cross-linked polyethylene, polyimide,ethylene vinyl acetate, styrene maleic anhydride, styrene-acrylonitrile,poly(methyl methacrylate), polytetrafluoroethylene, polybutylene,polylactic acid, polyvinylidene chloride polyvinyl chloride, polyvinylacetate, polyvinyl acetate co-polyvinylpyrrolidone,polyvinylpyrrolidone, polyvinyl alcohol, cellulose, lignin, celluloseacetate butryate, polypropylene, polypropylene carbonate, propylenecarbonate, polyethylene, ethyl alcohol, ethylene glycol, ethylenecarbonate, glycerol, polyethylene glycol, pentaerythritol, polyadipate,dioctyl adipate, triacetyl glycerol, triacetyl glycerol-co-polyadipate,tributyrin, triacetin, chitosan, polyglycidyl methacrylate, polyglycidylmethacrylate, oxypropylated glycerine, polyethylene oxide, lauric acid,trilaurin, citrate esters, triethyl citrate, tributyl citrate, acetyltri-n-hexyl citrate, saccharin, boron nitride, thymine, melamine,ammonium chloride, talc, lanthanum oxide, terbium oxide, cyclodextrin,organophosphorus compounds, sorbitol, sorbitol acetal, sodium benzoate,clay, calcium carbonate, sodium chloride, titanium dioxide, metalphosphate, glycerol monostearate, glycerol tristearate,1,2-hydroxystearate, cellulose acetate propionate, polyepichlorohydrin,polyvinylphenol, polymethyl methacrylate, polyvinylidene fluoride,polymethyl acrylate, polyepichlorohydrin-co-ethylene oxide, polyvinylidene chloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, and/or other similar materials. Inone embodiment, PHA, methanotrophic, autotrophic, and/or heterotrophicmicroorganism biomass, and a non-PHA polymer are mixed and meltedtogether. In one embodiment, the concentration of non-PHA microorganismbiomass in such a mixture ranges from 0.0001% to 90%, 0.1% to 30%, 0.1%to 10%, or 0.5% to 8%, and overlapping ranges thereof. In oneembodiment, the functional characteristics of a polyhydroxyalkanoate(PHA) material are augmented, controlled, or optimized, comprising thesteps of: (a) providing a PHA and a microorganism biomass, (b) combiningthe PHA and the biomass in a mixture to form a compound, (c) heating thecompound to between 40 degrees Celsius and 250 degrees Celsius. In oneembodiment, the biomass is present in said mixture at a concentration ofabout 0.1 to about 20%. In one embodiment, the biomass is present insaid mixture at a concentration of about 0.1 to about 80%. In oneembodiment, the biomass is present in said mixture at a concentration ofabout 0.1 to about 8%. In one embodiment, the biomass is methanotrophicbiomass. In one embodiment, the biomass is autotrophic biomass. In oneembodiment, the biomass is heterotrophic biomass. In one embodiment, thebiomass is present in said mixture at a concentration of about 0.1 toabout 20%. In one embodiment, the functional characteristics of apolyhydroxyalkanoate (PHA) material are optimized, controlled, oraugmented, comprising the steps of: (a) providing a PHA, a microorganismbiomass, and a non-PHA polymer, (b) combining the PHA, the non-PHApolymer, and the biomass in a mixture to form a compound, and (c)heating the compound to between 40 degrees Celsius and 250 degreesCelsius. In one embodiment, the biomass is present in said mixture at aconcentration of between about 0.1 to about 20%. In one embodiment, thebiomass is present in said mixture at a concentration of between about0.1 to about 80%. In one embodiment, the biomass is present in saidmixture at a concentration of between about 0.1 to about 8%. In oneembodiment, the biomass is methanotrophic biomass. In one embodiment,the biomass is autotrophic biomass. In one embodiment, the biomass isheterotrophic biomass. In one embodiment, the biomass is present in saidmixture at a concentration of between about 0.1 to about 20%. In oneembodiment, the biomass is present in said mixture at a concentration ofbetween about 0.1 to about 20%. In one embodiment, the biomass ispresent in said mixture at a concentration of between about 0.1 to about80%. In one embodiment, the biomass is present in said mixture at aconcentration of between about 0.1 to about 8%. In one embodiment, thebiomass is methanotrophic biomass. In one embodiment, the biomass isautotrophic biomass. In one embodiment, the biomass is heterotrophicbiomass. In one embodiment, the non-PHA polymer is one or more of thefollowing: polypropylene, polyethylene, polystyrene, polycarbonate,acrylonitrile butadiene styrene, polyethylene terephthalate, polyvinylchloride, fluoropolymers, liquid crystal polymers, acrylic,polyamide/imide, polyarylate, acetal, polyetherimide, polyetherketone,nylon, polyphenylene sulfide, polysulfone, cellulosics, polyester,polyurethane, polyphenylene oxide, polyphenylene ether, styreneacrylonitrile, styrene maleic anhydride, thermoplastic elastomer, ultrahigh molecular weight polyethylene, epoxy, melamine molding compound,phenolic, unsaturated polyester, polyurethane isocyanates, urea moldingcompound, vinyl ester, polyetheretherketone, polyoxymethylene plastic,polyphenylene sulfide, polyetherketone, polysulphone, polybutyleneterephthalate, polyacrylic acid, cross-linked polyethylene, polyimide,ethylene vinyl acetate, styrene maleic anhydride, styrene-acrylonitrile,poly(methyl methacrylate), polytetrafluoroethylene, polybutylene,polylactic acid, and/or polyvinylidene chloride, polyvinyl chloride,polyvinyl acetate, polyvinyl acetate co-polyvinylpyrrolidone,polyvinylpyrrolidone, polyvinyl alcohol, cellulose, lignin, celluloseacetate butryate, polypropylene, polypropylene carbonate, propylenecarbonate, polyethylene, ethyl alcohol, ethylene glycol, ethylenecarbonate, glycerol, polyethylene glycol, pentaerythritol, polyadipate,dioctyl adipate, triacetyl glycerol, triacetyl glycerol-co-polyadipate,tributyrin, triacetin, chitosan, polyglycidyl methacrylate, polyglycidylmethacrylate, oxypropylated glycerine, polyethylene oxide, lauric acid,trilaurin, citrate esters, triethyl citrate, tributyl citrate, acetyltri-n-hexyl citrate, saccharin, boron nitride, thymine, melamine,ammonium chloride, talc, lanthanum oxide, terbium oxide, cyclodextrin,organophosphorus compounds, sorbitol, sorbitol acetal, sodium benzoate,clay, calcium carbonate, sodium chloride, titanium dioxide, metalphosphate, glycerol monostearate, glycerol tristearate,1,2-hydroxystearate, cellulose acetate propionate, polyepichlorohydrin,polyvinylphenol, polymethyl methacrylate, polyvinylidene fluoride,polymethyl acrylate, polyepichlorohydrin-co-ethylene oxide, polyvinylidene chloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, and/or phenolpoly(4,4′-dihydroxydiphenyl ester.

Purifying the Isolated PHA

In some embodiments, isolated PHA is purified to produce a PHA materialthat is substantially pure PHA. In some embodiments, the isolated PHA ispurified to at least 20% pure PHA by dry weight. In some embodiments,the isolated PHA is purified to at least 55% pure PHA by dry weight,while in some embodiments, the isolated PHA is purified to at least 90%pure PHA by dry weight. In additional embodiments, purity of theisolated PHA is between about 90 and 99.9%, including about 91, about92, about 93, about 94, about 95, about 96, about 97, about 98, andabout 99% pure.

In several embodiments, isolated PHA may be recovered by any one, or acombination, of the methods described above, including, but not limitedto: washing, filtration, centrifugation, dewatering, purification,oxidation, non-PHA material removal, solvent removal, and/or drying. Insome embodiments, isolated PHA is recovered according to the manner inwhich it has been removed from the culture. For example, in embodimentsin which solvent-based extraction is employed, a recovery method may beemployed to remove the isolated PHA from the solvent and/or othernon-PHA material. In one embodiment, solvent may be used to extract thePHA, wherein the solvent is then substantially removed from the isolatedPHA by carrying out PHA precipitation and filtration, excess solventdistillation and/or removal, and/or drying, resulting in the recovery ofdry, isolated PHA. In embodiments employing enzyme, surfactant,protonic, hydroxide, and hypochlorite-based extraction techniqueswherein the dissolution of non-PHA material is induced, isolated PHA maybe filtered, washed, separated, centrifuged, and/or dried, resulting inthe recovery of dry, isolated, purified PHA. The resultant PHA, in someembodiments, is further used in downstream processing, includingthermoforming. In one embodiment, the concentration of PHA is caused togo high enough (e.g., above about 70%, about 80%, or about 90%) toinduce cell fragility and surprisingly cause PHA to be excreted orotherwise liberated extracellularly, through either no cell walltreatment or minimal cell wall treatment (such as, e.g., shear (inducedby a liquid pump or agitator), heat (included by water wherein thetemperature exceeds 10 degrees Celsius), or pH adjustment (low pH and/orhigh pH). In one embodiment, a method of causing the cell to becomefragile and excrete or enable the extracellular liberation or simplepurification of a PHA-biomass material by inducing a very highconcentration of PHA is employed to enable an avoidance of the usage oftoxic substances in purification or other processing steps, such aschlorine or chlorinated materials (such as chlorinated solvents, sodiumhypochlorite, chlorine gas, or other toxic chemicals. In one embodiment,Applicants have surprisingly discovered that by inducing a suppressionof an overproduction control switch or otherwise causing methanotrophicmicroorganisms, autotrophic microorganisms, and/or heterotrophicmicroorganisms to produce PHA at high concentrations (e.g., above about60%, above about 70%, above about 80%, and/or above about 90%), it ispossible to avoid the use of chlorine in purification or processingsteps—e.g., wherein the only chemicals and/or conditions required toproduce purified comprise: 1) water, 2) water and pressure, 3) water andcarbon dioxide, 4) water and shear, 5) water and heat, 6) heat, 7)shear, 8) liquid-solid separation, 9) solid-solid separation, 10)liquid-liquid separation, 11) spray drying, 12) sonication, 13)flocculation, 14) ultrasonic treatment, 15) lyophilization, 16) waterand hydrogen ions, 17) EDTA, 18) water and hydroxide ions, 19) sub orsuper critical carbon dioxide, 20) sub or super critical water, and/or21) a combination of these chemicals and/or conditions. In oneembodiment, PHA is separated from biomass or other non-PHA materials,including whole-cell microorganisms, enzymes, cell-surface attachedenzymes, isolated enzymes, and/or a combination thereof, and non-PHAmaterials are recycled for use as a catalyst, nutrient, carbon source,nitrogen source, nutrient source, mineral source, whole-cell catalyst,isolated enzyme, or other material for further polymerization, cellgrowth, or chemical synthesis. In one embodiment, water is removed fromor reduced in the PHA solution by using a batch, semi-batch,semi-continuous, and/or continuous water removal system, such as a spraydryer (including co-current, counter-current, pressure nozzle, rotarydisc, or otherwise), extruder (including single screw or twin screw),lyophilizer, filter (including filter press, pressurized filter, rotaryfilter, screw filter, anionic, cationic, and/or othermaterial-coagulating or polymer-assisted filter), flocculator, dissolvedair flotation mechanism, and/or a combination of each of these waterreducing mechanisms.

Returning PHA-Reduced Biomass to the PHA-Producing Culture to ConvertPHA-Reduced Biomass into PHA

In several embodiments of the invention, the PHA-reduced biomass isreturned to the culture to cause the biomass-utilizing microorganismswithin the culture to convert the carbon within the PHA-reduced biomassinto PHA. By using PHA-reduced biomass as a source of carbon for theproduction of microorganisms in a microorganism fermentation system, aseries of biochemical enzymatic pathways are generated in situ by themicroorganism culture to carry out the metabolic utilization ofPHA-reduced biomass for growth, reproduction, and PHA synthesis.

Without being limited by theory, it is believed that gas-utilizingmicroorganisms and biomass-utilizing microorganisms are able to co-existas a single microorganism consortium because they utilize sources ofcarbon that require distinctly different bioenzymatic assimilationpathways. For instance, while methane metabolism requires the methanemonooxygenase enzyme to catalyze the conversion of methane into methanolfor cellular assimilation, and methane monooxygenase is competitivelyinhibited by a wide range of compounds, it is not inherently deactivatedby high concentrations of cellular biomass, including PHA-reducedbiomass. Similarly, the chlorophyll-based metabolic assimilation systemsrequired for the conversion of carbon dioxide into biomass and PHA arenot inherently deactivated or competitively inhibited by highconcentrations of cellular biomass, including PHA-reduced biomass.Likewise, the enzymatic architecture enabling the metabolic utilization,breakdown, and/or assimilation of PHA-reduced biomass is not inherentlydeactivated or competitively inhibited by high concentrations of methaneand/or carbon dioxide, particularly as the process requires neithermethane monooxygenase nor chlorophyll. Without being limited by theory,Applicant believes that the relatively non-competitive, and in somecases commensal or mutualistic relationships between microorganismsconsuming a carbon-containing gas and a PHA-reduced biomass, make itpossible to create a microorganism culture comprising biomass-utilizingmicroorganisms and gas-utilizing microorganisms, wherein bothcarbon-containing gases and PHA-reduced biomass may be metabolized assimultaneously assimilable sources of carbon.

In the case of autotrophic, methanotrophic, and/or biomass-utilizingmicroorganisms, Applicant has found that a mutualistic,positive-feedback loop relationship can be created in a single (oroptionally multiple) reaction vessel. In such embodiments, the oxygencreated by autotrophic metabolism is utilized by methanotrophic and/orbiomass-utilizing microorganisms for metabolic functions, the carbondioxide created by methanotrophic and/or biomass-utilizing microorganismmetabolism is utilized for autotrophic metabolism, the methane and/orcarbon dioxide created by anaerobic methanogenic microorganisms isutilized by methanotrophic microorganisms, and the biomass created bymethanotrophic, autotrophic, and/or heterotrophic microorganisms is usedto provide a source of carbon to methanogenic and/or other heterotrophicmicroorganisms. Due to the microscopic-level induction of oxygen and/orcarbon dioxide created therein, mass transfer efficiencies in severalembodiments are significantly improved over traditional gas inductionmeans, such as gas sparging, mechanical mixing, static mixing, or othermeans known in the art. To applicant's knowledge, prior to thedisclosure herein, the use of autotrophic microorganisms cultured inassociation with heterotrophic microorganisms has never been suggestedas a means to improve mass transfer efficiencies, supply oxygen, and/oraugment microorganism growth rates in a positive feedback loop system.

In several further embodiments of the invention, PHA-reduced biomass isused by heterotrophic microorganisms, including acidogenic, acetogenic,and methanogenic microorganisms, to produce methane, which is furtherutilized by methanotrophic microorganisms to produce biomass, includingPHA. In some embodiments of the invention, anaerobic microorganismscoexist with aerobic microorganisms under microaerobic conditions (e.g.,mean dissolved oxygen concentrations approximately 0.00-1.0 ppm,including about 0.001 to about 0.002 ppm, about 0.002 to about 0.03 ppm,about 0.03 to about 0.04 ppm, about 0.04 to about 0.5 ppm, about 0.5 toabout 0.6 ppm, about 0.6 to about 0.7 ppm, about 0.7 to about 0.8 ppm,about 0.8 to about 0.9 ppm, about 0.9 to about 1.0 ppm, and overlappingranges thereof.

In some embodiments of the invention, heterotrophic, methanotrophic,methanogenic, and/or autotrophic microorganisms are divided intomultiple stages and vessels, in particular, anaerobic and aerobicstages, in order to carry out the conversion of PHA-reduced biomass intomethane and PHA. In further embodiments of the invention, PHA-reducedbiomass is returned to the culture using one or more vessels, whereby itis first converted to carbon dioxide, methane, and/or volatile organiccompounds by a heterotrophic, e.g., methanogenic, microorganismconsortium under anaerobic conditions and then converted to PHA bymethanotrophic microorganisms under aerobic conditions, whereby carbondioxide is also metabolized or otherwise used by autotrophicmicroorganisms, methanotrophic microorganisms, and heterotrophicmicroorganisms.

In several embodiments, light intensity is utilized to regulate thegrowth rate of heterotrophic and/or methanotrophic microorganisms. Insome embodiments, light intensity is manipulated to regulate thegeneration of oxygen by autotrophic microorganisms. In some embodiments,the rate of oxygen generated by autotrophic microorganisms issubsequently used to control the growth and metabolism of heterotrophicand methanotrophic microorganisms.

In several embodiments, carbon dioxide is supplied to autotrophicmicroorganisms in the form of carbon dioxide created by methanotrophicand/or heterotrophic microorganisms. In some embodiments, each of thesevarieties of microorganism is cultured in a single vessel. In someembodiments, methane, sugar, biomass, and/or another non-carbon dioxidesource of carbon is used to grow autotrophic microorganisms. Toapplicant's knowledge, autotrophic microorganisms have never beencultured using methane as a sole carbon input.

Some gas-utilizing microorganisms are unable to produce highconcentrations of intracellular PHA. However, according to severalembodiments, certain microorganism consortiums utilizing PHA-reducedbiomass, or derivatives thereof, as a source of carbon are able togenerate high intracellular PHA concentrations and thus effectivelyconvert low PHA concentration biomass derived from a carbon-containinggas into a high PHA concentration biomass material under the conditionsdisclosed herein. In several embodiments, the concentration (by weight)of intracellular PHA is between about 10% to about 30%, about 30% toabout 50%, about 50% to about 70%, about 70% to about 80%, about 80% toabout 90%, about 90% or more, and overlapping ranges thereof. Thus, inone embodiment, the culture is contacted with the PHA-reduced biomassand then manipulated, according to the processes described herein, toeffect PHA synthesis, wherein the PHA-reduced biomass is converted intoPHA by biomass-utilizing microorganisms. In some embodiments, PHAsynthesis is induced by nutrient limitation, nutrient excess, nutrientimbalance, or large shifts in nutrient concentration. In still furtherembodiments, PHA synthesis is induced by reducing the availability ofnitrogen, oxygen, phosphorus, or magnesium to the culture. In someembodiments, these nutrients are simultaneously reduced (to varying orsimilar degrees). In some embodiments, the nutrients are reducedsequentially. In some embodiments, only one of the nutrients is reduced.For example, in certain embodiments, PHA synthesis is induced byreducing the availability of oxygen to the culture. In some embodiments,this is achieved by manipulating the flow rate of air or oxygen into thegrowth medium. In some embodiments, manipulation of the flow rate ofother carbon-containing gases, such as methane and/or carbon dioxide,into the growth medium, or otherwise manipulating the rate of gastransfer in a system (e.g., by adjusting mixing rates or light injectionrates) is employed. In one embodiment, oxygen limitation is induced byreducing the flow rate of oxygen into the growth medium. In anotherembodiment, oxygen limitation is induced by reducing the rate of lighttransmission into the medium to reduce the production of oxygen byautotrophic microorganisms. In some embodiments of the invention, theconcentration of PHA generated in a biomass-utilizing microorganismculture utilizing PHA-reduced biomass as a source of carbon is at least5%, at least 20%, or at least 50% by dry cell weight; in particularlypreferred embodiments of the invention, the concentration of PHA in abiomass-utilizing microorganism is at least 80% by dry cell weight.

In some embodiments, a PHA-reduced biomass recycling system is utilizedwherein substantially all (e.g., at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 98%) of the PHA-reducedbiomass produced is contacted with the culture until it is convertedinto PHA. In some such embodiments, solid sources of carbon aresubstantially output from the process or culture only in the form ofisolated PHA.

In several embodiments, as carbon-containing gases are continually addedto the medium to effect the production of biomass, the process disclosedabove is repeated. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the culture is removed from the medium,PHA is extracted from the PHA-containing biomass, PHA-reduced biomass isseparated from isolated PHA, isolated PHA is recovered, purified, anddried, and PHA-reduced biomass is sent back to the culture and convertedby the culture into PHA. In one embodiment, substantially allPHA-reduced biomass produced is contacted with the culture until it isconverted into isolated PHA, and solid sources of carbon aresubstantially output from the process only in the form of isolated PHA.In other embodiments, carbon is substantially output from the systemonly in the form of PHA and methane, carbon dioxide, and/or volatileorganic compounds.

The following example is provided to further illustrate certainembodiments within the scope of the invention. The example is not to beconstrued as a limitation of any embodiments, since numerousmodifications and variations are possible without departing from thespirit and scope of the invention.

Example 1

A fermentation system comprising one or more vessels are partiallyfilled with one or more liquid growth mediums, wherein the mediumcomprises methanotrophic, autotrophic, methanotrophic, and/or otherheterotrophic or biomass-utilizing microorganisms containing PHA, and,per liter of water, 0.7-1.5 g KH₂PO₄, 0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃,0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28 mg CaCl₂*2H₂O, 5.0-5.4 mg EDTANa₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O, 0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mgMnCl₂*2H₂O, 0.06-0.08 mg ZnCl₂, 0.05-0.07 mg H₃BO₃, 0.023-0.027 mgNiCl₂*6H₂O, 0.023-0.027 mg NaMoO₄*2H₂O, 0.011-0.019 mg CuCl₂*2H₂O. Oneor more of the mediums are anaerobic and/or aerobic, and carboncontaining gases, including methane, carbon dioxide, and volatileorganic compounds, as well as optionally air or oxygen, are fed into allor part of the system to induce the growth and reproduction ofmicroorganisms through the utilization of carbon-containing gases, aswell as the production of PHA.

Next, a portion of the media volume of the fermentation system is passedthrough a basket centrifuge to increase the solids content of the mediumto approximately 167 g/L. The solids-containing centrate phase of thecentrifuged solution is then transferred to a PHA extraction vessel, andthe substantially solids-free filtrate phase of the centrifuged solutionis recycled back to the fermentation system.

In some embodiments, the solids-containing centrate phase is optionallychemically pre-treated prior to extraction. In some embodiments, one ormore of acids, bases, chloride, ozone, and hydrogen peroxide is added.In several embodiments, chemical pre-treatment increases the efficiencyand yield of the subsequent extraction process. In some embodiments, thechemical pre-treatment functions to break down the cell well (partiallyor fully), thereby liberating a greater portion of the intracellularPHA. In some embodiments, chemical pre-treatment dissolves and/orremoves impurities that negatively impact the PHA extraction process. Insome embodiments, chemical pre-treatment enhances cell agglomeration,which increases the percentage of microorganisms that are extracted(e.g., cells in an agglomerated mass are not separated or lost intransfer steps). Next, following optional chemical pre-treatment,solvent is added into the PHA extraction vessel to create a solventsolution, and the solvent solution is then mixed for a period of time tocause the PHA in both the microorganisms to dissolve into the solvent,and thereby create PHA-rich solvent and PHA-reduced biomass. Over thecourse of a defined mixing period (e.g., 0.1-10 hours), the PHA contentof the microorganisms is reduced by about 80% as it is dissolved intothe solvent.

Next, the solvent solution comprising the PHA-rich solvent andPHA-reduced biomass is passed through a filter located at the bottom ofthe PHA extraction vessel, and the PHA-rich solvent is thereby separatedfrom the PHA-reduced biomass. Water is then added to the PHA extractionvessel to create a water-biomass solution, and the water-biomasssolution is then heated to 75° C. to cause any remaining solventassociated with the PHA-reduced biomass to exit the PHA extractionvessel as a gaseous vapor. The vapor discharged from the PHA extractionvessel is then passed through a heat exchanger and recovered as liquidsolvent. Meanwhile, the PHA-rich solvent is transferred to a PHApurification vessel and mixed with room temperature water to create awater-solvent solution. The water-solvent solution is then heated tocause i) the solvent to exit the PHA extraction vessel as a gaseousvapor and ii) the isolated PHA to precipitate into the water and/orbecome a solid. The solvent vapor created by heating the water-solventsolution is then passed through a heat exchanger and converted intoliquid solvent.

The isolated PHA is then substantially dewatered by filtration in aNutsche filter, and the Nutsche filter containing the substantiallydewatered isolated PHA is then heated to remove any additional volatilecompounds, including trace water and/or solvent. Following heat dryingin the Nutsche filter, the isolated PHA is recovered as substantiallypure PHA (e.g., greater than about 90% PHA).

Concurrently, the water-biomass solution comprising PHA-reduced biomassand water is transferred from the PHA extraction vessel back into thefermentation system, where the PHA-reduced biomass is contacted with oneor more of the microorganism cultures. Next, the medium of thefermentation system is manipulated to cause the one or moremicroorganisms within the system to metabolize the PHA-reduced biomassas a source of carbon and nutrients.

Depending on the embodiment, the culture conditions are adjusted todetermine the point at which the inception of the growth or PHAmetabolism phase occurs. As discussed herein, manipulation of theconcentration of one or more growth culture media nutrients can alterthe metabolic pathways favored by certain microorganisms. Additionally,the use of PHA-reduced biomass-derived carbon for the production ofadditional biomass versus the production of PHA can be tailored based onwhether the intent is to grow the culture (e.g., increase the overallbiomass) or to harvest PHA (e.g., shift the culture from growth phase toproduction of PHA). As such, the reduction, increase, or adjustment ofthe concentration certain growth nutrients, and the timing of suchadjustment, plays a role in the metabolic state and PHA production ofthe culture. Adjustment of growth nutrients can occur at any point afterthe PHA-reduced biomass is returned to the microorganism system. In someembodiments, adjustment is immediate (e.g., within minutes to a fewhours). In some embodiments, a longer period of time elapses. In someembodiments, adjustment in one or more growth nutrients occurs afterabout 2 to 4 hours, after about 4 to 6 hours, after about 6 to 8 hours,after about 8 to 10 hours, after about 10 to 12 hours, after about 12 to14 hours, after about 14 to 18 hours, after about 18 to 24 hours, andoverlapping ranges thereof. In still additional embodiments, adjustmentin one or more growth nutrients occurs after about 2 to about 5 days,about 5 to about 10 days, about 10 to about 15 days, about 15 to about20 days, about 20 to about 30 days, about 30 to about 50 days, andoverlapping ranges thereof. In some embodiments, longer times elapseprior to adjusting one or more growth nutrients to induce PHApolymerization.

After a desired period of time has elapsed, the dissolved oxygen and/ornitrogen concentration (or concentration of another nutrient) of one ormore parts of the medium is reduced or adjusted to cause one or more ofthe microorganisms within the system to utilize the PHA-reduced biomassin the medium as a source of carbon for the synthesis of PHA. In someembodiments, the percent adjustment ranges from about 20% to about 100%,including about 20% to about 30%, about 30% to about 40%, about 40% toabout 50%, about 50% to about 60%, about 60% to about 70%, about 70% toabout 80%, about 80% to about 90%, about 90% to about 100%, andoverlapping ranges thereof. In several embodiments, depending on thecharacteristics of a culture in a given embodiment, a specificpercentage reduction, increase, or adjustment in nutrient may not benecessary, but a reduction, increase, or adjustment is used that issufficient to convert certain cells from a relative growth phase to arelative PHA synthesis phase. After approximately 12-24 hours of PHAsynthesis, substantially all of the PHA-reduced biomass within thegrowth medium has been metabolized into biomass-utilizing microorganismbiomass, including PHA. In certain embodiments, greater or lesser PHAsynthesis times result in varying percentages of the PHA-reduced biomasswithin the growth medium being metabolized into biomass, including PHA.

As carbon containing gases are continually added to the fermentationsystem to effect the production of biomass, the process is repeated,wherein solid sources of carbon substantially exit the system only inthe form of PHA. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the fermentation vessel is passedthrough a dewatering centrifuge to increase the solids content of thePHA-containing biomass, PHA is extracted from the removed PHA-containingbiomass using a solvent-based extraction system to create PHA-reducedbiomass and isolated PHA, PHA-reduced biomass is separated from isolatedPHA, isolated PHA is recovered, purified, and dried, and PHA-reducedbiomass is sent back to the fermentation system and converted bymicroorganisms into PHA, such that substantially all PHA-reduced biomassproduced is contacted with the culture until it is converted intoisolated PHA, and wherein solid sources of carbon are substantiallyoutput from the process only in the form of isolated PHA.

While the above description of several compositions, systems, andmethods contains many specificities, it should be understood that theembodiments of the present invention described above are illustrativeonly and are not intended to limit the scope of the invention. Numerousand various modifications can be made without departing from the spiritof the embodiments described herein. Accordingly, the scope of theinvention should not be solely determined by the embodiments describedherein, but also by the appended claims and their legal equivalents.

What is claimed is:
 1. A method for enhancing polyhydroxyalkanoate (PHA)generation in a methanotrophic culture by reducing copper concentrationsto effect particulate methane monooxygenase (pMMO) production, themethod comprising: (a) contacting a culture of methanotrophicmicroorganisms with a medium comprising copper, one or more additionalnutrients, and a carbon-containing gas that can be metabolized by saidculture; (b) incubating said culture in said medium to cause growth ofsaid culture; and (c) inducing a selection pressure in said culture totransform said culture into a culture that generates PHA preferentiallythrough pMMO by: (i) reducing the concentration of copper in said mediumto cause production of soluble methane monooxygenase (sMMO) and/orparticulate methane monooxygenase by said culture, wherein saidconcentration of copper causes the production of sMMO in somemethanotrophic microorganisms; (ii) reducing the concentration of one ormore said nutrient in said medium to cause said culture to generate PHAfrom said carbon-containing gas using said sMMO or said pMMO, whereinPHA is generated by pMMO at a greater rate as compared to sMMO, (iii)returning the culture to the growth conditions of step (b), whereinmicroorganisms having higher intracellular concentrations of pMMO andPHA grow at a greater rate as compared to those with lower intracellularpMMO and PHA concentrations; and (iv) repeating steps (ii) and (iii),wherein said repetitions selectively favor growth of microorganisms thatproduce PHA via pMMO, thereby facilitating the pMMO-mediated productionof PHA at reduced copper concentrations, and resulting in a culturecomprising essentially only microorganisms that use pMMO to produce PHA.